GRE BioChem

8/12/2019 GRE BioChem

http://slidepdf.com/reader/full/gre-biochem 1/33

TO THE GRE BIOCHEMISTRY SU JECT TEST

n ~ g Marquart

CHEMISTRY

electron donors

electron acceptors

law of thermo energy converted, never created or destroyed

law of thermo - universal entropy always increases

projection - 2D representation of 3D structure

- same chemical bonds

mirror image steroisomers

- steroisomers that are not mirror images

- clockwise up

- counterclockwise up

- cyclic rotation about 1 axis),

dihedral rotation along 2 axis),

icosahedral- 20 faced (virus capsid), helical

energy for synth on growing polymer

proteins & fatty acids

energy for synth on added monomer

DNA, RNA, polysaccharides

lattice of one molecule trapping another

layer shell of water around hydrophobic molecule

- structural motif that binds NAD/NADP cofactors

> [NADHj

nicotinamide adenine dinucleotide

oxidations, catabolic

< [NADPH]

nicotinamide adenine dinucleotide phosphate

reductions, anabolic

flavin adenine dinucleotide

- rapid oxidation & reduction

heme - porphyrin ring + (held by 4 nitrogen)

proteins iron-sulfer center)

Q) 1 2 electrons

- from vitamin riboflavin

expresses relative contrib. of enzymes to

metabolites flow through pathway. sum of lUX control

ical catalysts

different forms of enzyme that catalyze same reaction

does not contain easily ionizable functional groups

catalytic antibody

Scatchard analysis - quantifies affinity blw receptor & ligand

TP resonance & charge on Pi contribute to high potential

phosphorylation - usually at AAs w i OH side chains (5er, Thr, Tyr)

Inhibition

Agonists - structural analogs that bind receptor & mimic effects

Antagonists anologs that bind wlo triggering normal effect

competitive inhibition - binds active site, blocking catalysis

o Vmax same, Km increases plots cross)

un competitive inhibition - bind to E5 complex at separate si

o Vmax & Km lowered plots parallel)

mixed inhibition - binds alternative site wi or wlo substrate

o effects both Vmax & Km

noncompetitive inhibition very unusual

. 0 Vmax lowered, Km same (plo ts cross)

Alloseteric Enzymes

heterotropic effects non-substrates shift sigmOidal plot to right

homotropic effects - substrates shift sigmoidal plot to left

i c h a e l i s ~ e n t e n equation

hyperbolic dependence of Va on [51

usefulfor detecting

allostericregulation

neither Vmax nor Km immediately clear

double-reciprocal plot 1/V vs 1/[5])

more accurate determination ofV max & Km

o y-intercept - l Vmax

ox- intercept l /Km

Vmax - reaction rate when all active sites saturated wi substrate

m Michaelis constant) - [5] at]l, Vmax

Keat general rate constant / turnover number) of substrate

converted to product when saturated per unit time (Vmax [enzym

eal m - specificity constant

Q mass-action ratio) - [P]/[Rj

1-K,n

ENZYMES

zymogen proenzyme - inactive enzyme precursor

Condensation Reactions

synthases - catalyze condensation wlo NTP

synthetases - catalyze condensation wi NTP

Jigases catalyze condensation of two atoms wi NTP

hydroxylase - hydroxylates substrate



-1-2 oxygens of O2 incorporated into substrate

- both atoms of O2 incorporated into substrate

involved in nearly all aromatic ring cleavages

/ oxioreductases - catalyze redox reactions

- hydrolytic cleavage

cleave tryglycerides into glycerol & fatty acids

- interconvert stereo, structural or positional isomers

mutase - transfers functional group win single molecule

serine/threonine kinases - primarily pp-ate serine

tyrosine kinases - Src largest family of cytoplasmic TKs

function oxidase - incorporates O2 into steroids, etc

non-protein bound to enzyme

2+ - complexes w/ ATP to form substrate for kinases

2+ - often found in enzyme active site

- permanently bound cofactor

nucleotide NAD, etc

cofactor in coenzyme A, NAD+, FAD, NADP+

- organic non-protein carries groups bin enzymes

- coenzyme in carboxylation reactions (C0 2 carrier)

coenzyme-like carrier of hexose groups

- protein kinase A (cAMP-dep.) - serine/threonine A-kinases

protein kinase B (AKT)

- protein kinase C (Ca2+-dep.)

- binds to cytoplasmic face of PM

- stim'd by Ca2+ & diacylglycerol (DAG)

- atypical PKCs - not activated by Ca2+ & DAG (diacylglycerol)

synthesis

Precursors AA Details

a - k e t o g l u h ~ + ~ glutamate amination

PKG - protein kinase G (cGMP-dep.)

PKR - protein kinase R (dsRNA-dep.)

AMPK AMP-activated protein kinase

metabolic master switch regulating glucose uptake,

oxidation, biogenesis of GLUT4 and mitochondria

Vitamins \ .t' 1 tit uw-\.oAI i\ I >'VVSVitamin B6 {Pyndoxal phosphate (PLP))

cofactor for aminotransferase b/w glutamate & other AA

Vitamin B 2 (cobalamin) - transfer methyl groups (methionine syn

Fat solubleVitamin A (retinal)

regulates gene expo in epithelial tiss., rod & cone response

. retinoic acid activates hox genes

Vitamin D3 (cholecalciferol) - converted to 1,25-

dihydroxycholecalciferol, regulates Ca2+ uptake & maintenance

Vitamin E (tocopherols) - destroys oxygen radicals

Vitamin K

essential for prothrombin production

essential for glutamate y-carboxylation

Vitamin Coenzyme Reaction

Thiamine (B1) Thiamine Transfer of aldehydpyrophosphate

Riboflavin (B 2) FADH Redox

I Niacin NADH/NADPH Redox

Pantothenic acid Coenzyme A Transfer of acyl gro

Pyridoxine{Bb Pyridoxal phosphate-¥l{ Amino acid activatio

Biotin (H/B7) Biotin Transfer of CO2

Lipoic acid Upoamide Redox, acyl activatio

Folic acid Tetrahydrofolate Transfer carbon gro

Cobalamin I Transfer methyl gro

MINO ACIDS PROTEINS

AA characteristics

ionization

in solution, AAs act as zwitterions (dipolar ions)

isoelectric point pI) - pH where molecule has no net charge

Enzyme/lnterm Cof

glutamate dehydrogenase PLP

(glutamate)#glutamine reg'ed by adenylation glutamine synthetase

#proline, #arginine

oxaloacetate aspartate transamination PLP

(aspa rtate) asparagine, *threonine, *Iysine a s p a r t y l - ~ - A M P + glutamine 7 asparagine

rtate + cysteine *methionine

#serine

#glycine serine hydroxymethyltransferase H4folate

(serine)#cysteine , , , , , + If id, ~ I

serine + methionine ( cystathionine adoMet

alanine-- transamination by PLPpyruvate

*valine, *Ieucine, *isoleucine • valine leucine - 2

PEP *phenylalanine

+ tyrosine chorismate - branch point phenylalanine hydroxylase

*tryptophani

PRPP *histidine i adenine + riboseI



pyridoxal phosphate PLP/Vitamin B6)

o aminotransferase b/w glutamate & other AAs

tetrahydrofolate HJolate) - transfers 1 carbons

o dihydrofolate reductase -dihydrofolate-7tetrahydrofolate

S-adenosyl methionine adoMet) - transfers methyls

starvation

stringent response regulation coordinated w/ [AA]

uncharged tRNA bind ribosomes

stringent actor binds ribosome

stringent factor catalyzes formation of ppGpp & pppGppppGpp binds rRN\ reducing synth

9 \l\. >

degradation

s converted to glucose & lipids

amino transferred to a-ketoglutarate (glutamate)

branched-chain a-keto acid dehydrogenase complex

o in extrahepatic tissues (muscle, adipose, kidney, & brain)

o unbranches leucine, isoleucine & valine

non-branched-chain amino acids degraded in liver

o aminotransferase that converts branched-chain amino

acids to a-keto acids absent in liver

Degraded Ipyruvate tryptophan, threonine , alanine, serine, cysteine, glycine I

acetyl-CoA phenylalanine, tyrosine, tryptophan, isoleucine, threonine i

(leucine, lysine (only ketogerifcf - ketogenic)

-ketogluta rate5 carbon skeletons) proline, glutamate, glu tamine

(converted to 5 carbons) arginine, histidine

Fumarate phenylalanine, tyrosine

succinyl-CoA threonine, methionine,d:s01eucine, val in?)

Oxaloacetate aspartate, aspargine

non-protein biomolecules

nitrogen donors in variety of biosynthetic reactionsy-aminobutyrate (GABA)

porphyrins (4 porphobilinogens)

+ arginine & methionine

o phosphocreatine - energy buffer in skeletal muscle

+ glutamate & cysteine

o GSH glutathione) - redox buffer

cotecholamines - dopamine, norepinephrine, epinephrine

melanin (tyrosine 3-monooxygenase (tyrosinase))

+ phenylalanine

o lignin (cell walls of plants)

auxin - plant growth hormone

serotonin

niacin (vitamin in NAD/NADP)

nitric oxide (NO)

histamine (decarboxylated)

Types

single secondary structure (support, shape, protection)

multiple secondary structure (enzyme & regulatory)

- stable, partiall y folded protein state

I

I

I

protein disulfide isomerase POI) - shuffles disulfide bonds

peptide prolyl cis-trans isomerase PPI) - converts cis & trans isom

BiP Binding immunoglobulin protein) binds translocated protei

in ER lumen, preventing aggregation or misfolding

Chaperones

Hsp (heat-shock proteins)

o Hsp60 - barrel shaped-chaperonin isolation chamber

o Hsp70 - BiP (in ER) assists folding during synth

ER chaperone proteins - calnexin & colreticulin

heat-shock response - trigged by accum. of misfolded proteins

unfolded protein response - trigged by accum. of misfolded prote

stims increased transcription of ER chaperones

slows protein production

Pr6tein Structure

peptide bonds - usually planar (trans rather than cis config)

Amino acid backbone

o Ca.-N - {J (phi) rotations

o Ca.-C - i /J (psi) rotations

o C-N bond in backbone is rigid

Ramachandran plot - allowed values for {J & i /J

Secondary structures most common aas)a helix (glutamate, metheonine, alanine)

o normally right-handed

sheet (isoleucine, valine, tyrosine)

connect antiparallel Bsheets (glycine, proline, & asparagine

o 8 turn - 4 residue connection

o V urn - 3 residue connection (considerably less commo

Supersecondary structures

8-a-8 loop, a-a corner, a/B barrel (8-22 B sheets)

Targeting signals

N-terminal sequences

targets for chloroplasts, rough ER, mitochon. (usually cleav

Internal sequences

signal patch multiple internal AAs seqs that form 3D signa

nuclear localization signal- targets protein for nucleus

o located almost anywhere not cleaved)

Protein Glycosylation

precursor oligosaccharide - preformed sugar (14) transferred en

Dolichol-lipid molecule that holds precursor oligosacchari

oligosaccharyl transferase - next to translocator Sec61 complex

N-/inked - connected to NHl on asparagines

complex accessible to modifications

high-man nose unmodified - held close to protein

O-Iinked - on OH (serine, threonine, hydroxylysine, hydroxyprolin

[less common]

Transmembrane proteins

hydropathy plots ID membrane-spanning helices (20-30

Ms)

most glycosylated (sugars added in ER & Golgi)

Protein regulation I modification

Modification TargetAA Change

Phosphate Ser, Thr, Tyr Proteins complexing, on/o

Methyl Lysine Histone code



Acetyl Lysine Histone code

,Ubiquitin Lysine mono - regulates transport,

poly - degradation

Palmityl Cysteine

=associate w/ membrane

for degradation by proteosomes

- proteasome core contains several proteases

triad -- serine, aspartate, histidine

found inside active site of proteases

- binding sites of complexed n z y ~ sPTB - phosphotyrosine-binding

Src homology - SH2 SH3

o SH3 - binds to proline-rich stretches of polypeptide

(TAGs) (3 fatty acids + glycerol)

derived from phosphatidate (diacylglycerol 3-phosphate)

o 1,2-diacylglycerol intermediate

depots of metabolic fuel (lipid droplets, bud off ER)

primary fuel during fasting converted to ketone bodies)proteins - last cellular material to be used as energy

~ 7 S offatty acids from triacylglycerols recycled

holipids / phosphoglycerides (phosphate + glycerol +

phosphatidylcholine (lecithin) - primary phospholipid

o phosphatidic acid + choline choline + ATP -)

• choline-Pi + CTP -) COP-choline

o methylation of phosphatidylethanolamine (in yeast)

phophatidylserine negative charge

o serine + COP-diacylglycerol

o derived from phosphatidylethanolamine (in mammals)

phosphatidylethanolamine

o (decarboxylation of phosphatidylserine)

important phospholipid in myelin heart tissue

glycerophospholipids w/ acyl chain in place of glycerol

sphingosine instead of glycerol

sites of biological recognition (define human blood groups)

ceramide - basic structural unit of sphingolipids

o fatty acid - amide linkage - sphingosine

phosphatidylcholine + ceramide

only non-glycerol membrane phospholipid

protect membrane

important for cell recognition/adhesion

o cerebrosides myelin (galactose reSidue)

o gangliosides - contain NANA (sialic acids) negative charged

o galactosides neutral charge?

- predominant membrane lipid in plant cells

Fatty acid synthesis

Location cytosol of higher eukaryotes, chloroplasts in plants

Details

CO2 or HCO r ) needed for first step

acetyl groups transferred across mito.memb. as citrate

NADPH used from citrate leaving mito pentose i pathway

fatty acyl grows by 2C from malonate (C02 lossed each step)

final product palmitate (16 C or stearate (18 C)

fatty acid synthetase complex - synth from acetyl-CoA

polypeptide w/ 6 enzyme activities + acyl carrier protein AC

. acetyl-CoA + CO2 - malonyl-ACP + acetyl -) acetoacetyl-ACP + CO

acetyl-CoA carboxylase - rate-limiting step

acetyl-CoA + CO 2 - malonyl-CoA

malonyl-CoA-ACP transferase (MT) - malonyl-CoA -) acetoacetyl-A

Squalene

3 0 ~ C l i n e a r precursor of cholesterol (6 isoprene units)

Synthesis

(1) 3 acetates -) mevalonate (6-C)

(2) mevalonate -) activated isoprenes(3) polymerization of six S-C isoprenes to squalene

HMG-CoA reductase - rate-limiting enzyme in mevalonate pathw

Cholesterol

steroid nucleus - 4 fused rings; 3 w/6-carbons 5-carbon

principle sterol (alcohol w/ OH at C-3) in mammals

lesser amounts in plants fungi (not in chloroplast memb)

OH of cholesterol steroids from O2

decreases permeability

high concentrations in lipid raft, endocytic membranes

Cholesterol Synthesis

(4) 3 isopentenyl pyrophosphate (5-carbon) condense to 15 car

(S) 2 is-carbon combine to 30-carbon molecule

(6) 30 carbon cyclized to form cholesterol

Major synthesis sites - ER of liver small intestine

Biomolecules derived from Cholesterol

vitamin D (essential control of calcium metabolism)

bile acids - aid in digestion of lipids (conjugated by glycine)

steroid hormones

glucocorticoids, mineralocorticoids, sex hormones

Fatty Acid Cholesterol TransportStructure

apolipoproteins, phospholipids, triacylglycerols cholestero

chylomicrons

carry triacyiglycerols from intestine to adipocytes for storag

VWL very low density lipop.)

carry cholesterol esters, triacylglycerols (from liver to tissue

LOL low density /ipop.)

lipoprotein lipase degrades TAGs, converting VLDL to LDL

transports cholesterol throughout body

taken up by endocytosis B-l00 apolipoprotein)

HDL high density lipop.)

removes cholesterol from blood, carrying i t to liver



degradation

-7 D-glyceraldehyde 3-phosphate -7 glycolysis

generate acetyl-CoA from fatty acids

animals cannot convert fatty acids into glucose b/c degradation

thru CCA does not lead to increase in oxaloacetate

a oxidation - in peroxisomes when ~ c a r b o n is methylated

oxidation - in mito. matrix perox., cyclegen 2 I\lADH,

w oxidation - in liver/kidney ER, cycle gen 1 NADH 1 FADH2

epimers - sugars that differ in config around 1 carbon only

anomers - isomeric form that differ at anomeric carbon

- a-isomer - OH in axial position (down)

- ~ i s o m e r - OH in equatorial position (up)

mutarotation - interconvertible thru noncyclic intermediates

reducing end - end w/ free anomeric carbon w/o glycosidic bond)

nonreducing - sugar w/ O-glycosidic bond on anomeric carbon

aldose - carbonyl group at end of chain (forming an aldehyde grou

- hemiacetal cyclic form of aldose

- aldonic acids - oxidation of carbonyl/anomeric carbon- uronic acids - oxidation of C6 carbon

Breaks carbon bond carbon - 2nd

C from carboxyl C-

3)]

ketose - carbonyl group internal

perox. mito. in mammals, only peroxisomes in yeast/plants

malonyl-CoA shuts down ~ o x i d a t i o no prevents futi le cycle (simul. fatty acid synth

oxidation)

enoyl-CoA isomerase - removes cis double bonds

u nsatu rated)

gens 1 NADH, 1 FADH 2 1 acetyl-CoA per cycle

odd-number fatty acids - propionyl-CoA

-7 methylmatonyl-CoA -7 succinyl-CoA -7 CCA

acyl-CoA dehydrogenase

o removes H a C, forming trans double bond

enoyl-CoA hydratase

o adds H20 across double bond

3-L-hydroxyacyl-CoA dehydrogenase

o converts hydroxyl to keto

acyl-CoA acetyltransferase (B-ketothiolase)

o thiol group cleavage by CoA

shuttle

carnintine transports fatty acyls <::14 C thru mito. inner

memb.

acyl-CoA -> fatty acyl-carnitine -> fatty acyl-CoA

long fatty acyl-CoA synthetase

o CoA + fatty acid -7 fatty acyl-CoA

carnitine acyltransferase I

carnitine-acylcarnitine translocase

o (acyl-carnitine/carnitine transporter )

carnitine acyltransferase

carbon - carbonyl carbon in carbohydrate ring structure

- mirror image stereoisomers

- non-mirror image stereo somers

substrate product Enzyme

glucose glucose 6-phosphatehexokinase

ATP ADP

G6P fructose 6-phosphate phosphoglucose isomerase

F6P fructose l,6-bisphosphatephosphofructokinase-l

ATP ADP

- hemiketal cyclic form of ketose

pyianoses - 6 membered carbon ring

furanoses - 5 membered carbon ring

Naming 5 -6

2 0 : 4 ~ 5 , 8 , 1 1 , 1 4 ) - 20 Carbon chain, double bonds at 8-9, 11-12, 14-1

Non-Glucose Sugars

lactose -7glucose -7 glucose 6-phosphate

galactose -7 glucose 1-phosphateglucose -7 glucose 6-phosphate

sucrose -7fructose -7

fructose 6-phosphate or

glyceraldehyde 3-phosphate

man nose -7 fructose -7fructose 6-phosphate or

glyceraldehyde 3-phosphat

trehalose -7 glucose -7 glucose 6-phosphate

dextrans - bacterial + yeast polysaccharides

GLUCOSEP THVV YSGlycolysis/gluconeogenesis regulation (substrate/futile cycle)

- fructose 6-phosphate <-(FBPase 1) / (PFK-1)-> fructose 1

bisphosphate

fructose 2,6-bisphosphate F2,6BP)

- regs glycolysis gluconeogenesis thru PFK-1

- (activates glycolysis) FBPase-1 (inhibits gluconeogenesis)

- made/broken down by PFK-2/FBPase-2

0 protein w/ 2 enzymatic functions

0 regulated by glucagon insulin controls thru cAMP

Inhibits ActivatesEnzyme

Directly Indirectly Directly Indirectly

PFK-l ATP, citrate F2,6BP Pi

FBPase-l(l,6) F2,6BP AMP triose p h o s p hPFK-2 triose phos phates Pi insulin

FBPase-2 2,6) F6P insulin

Inhib Stim irrev

G6P NADP/NADPH? pentose Pyes

p ,f:i1lA C

ATP, citrate ADP/AMP, F2,6PYes



~ dihydroxyacetone phosphatealdolase

glyceraldehyde 3-phosphate

DHAP GADP/G3P triose phosphate isomerase

GADP/G3P 1,3-bisphosphoglycerateGADP/G3P dehydrogenase

. NAD+ + Pi NADH

1,3BPG 3-phosphoglyceratephosphoglycerate kinase

ADP AlP

3PG 2-phosphoglycerate phosphoglycerate mutase

2PG phosphoenolpyruvate enolase

PEP pyruvate

ADP AlPpyruvate kinase

\

mainly from AAs from degraded proteins

primarily in liver (smaller extent in renal cortex)

2 pyruvate -> glucose (consumes 4 ATP, 2 GTP, & 2NADH)

bypass irreversible steps (1,3, & 10)

1 t

high nrg compound

ATP, alanine, acetyl-CoA,

long-chain fatty acidsyes

glucose 6-phosphatase removes Pi from G6P

o in ER of kidney & liver only

o compartmentalized to prevent abortion of glycolysis

Glycogenesis (glucose glycogen)

1 glucose + ATP glucose 6-phosphate + ADP

G6P -(glucose 6 - p h o s p h a t a s e ) ~ glucose + Pi

[glucose-1-phosphate + UTP UDP-glucose + pyrophosphate]

glycogen al-4Iink, al-6 branches every 8-12 residues

F6P + ATP fructose 1,6-bisphosphate + ADP

2: F1,6BP -(FBPase-1H F6P + Pi

phosphoenolpyruvate + ADP -> pyruvate + ATP

1: OXAc -(malate d e h y d r o g e n a s e ) ~ malate OXAc

allows transported out of mito.

GTP -(PEP c a r b o x y k i n a s e ) ~ PEP + C02 + GDP

glucose level

Lowers - insulin Ci+ channels triggers release from pancreas)

o glucose uptake, glycolysis, synth of fatty acids

o inhibits fatty acid mobilizati on (glycogen & TAGs storaged)

Raises - epinephrine glucagon cortisol

o gluconeogenesis in liver (inhibit glycolysis)

o glycolysis in muscle

o mobilizes fatty acids from triacylglycerols in adipose

transporters

UT4 - adipose & muscle, sequestered in absence of insulin

-liver, pancreas, hypothalamus, intestine (reabsorbs glucose)

glycogen phosphorylase phosphorylates nonreducing sugar

until 4 sugars upstream from branch

o glycogen phosphorylase alb a-active b-Iess

o controlled by pp-tion by phosphorylase kinase

o stim AMP

o inhib: ATP, glucose (binds to phosphorylase a b)

debranching enzyme

o transferase trans 3 sugars prior to branch to main strand

o glucosidase hydrolyzes branched glucose

phosphoglucomutase G1P glucose 6-phosphate

Enzyme

glycogen synthase lb (active /inactive)

o adds glucose to glycogen (requires 8 glucose chain)

glycogenin synth glucose primer for glycogen synthase I

o inhib by glycogen synthase kinase GSK-3) [3x pp d] rrprimed w i 1 pp by casein kinase II (CKII)

EnzymeInhibits Activates

Directly Indirectly Directly Indirect

phosphoprotein insulin

phosphatase 1

protein kinase A cAMP epin.

protein kinase B insulin

phosphorylase PP-1 PKA Ca +

kinase a/b

glycogen PP-1.r AlP n' - i phosphorylase peptin, ep

phosphorylase a/b kinase, AMP2+

glycogen synthase PKB insulin

kinase 3

glycogen synthase GSK-3 epin., C? PP-1, casein

kinase 2

CITRIC CID CYCLE Krebs)Pyruvate dehydrogenase complex - pyruvate N A D + - N A D H ) ~ C

+ acetyl-CoA

3 enzymes - 5 cofactors

o (24) Pyruvate dehydrogenase -thiamine

pyrophosphate TPP)

o (24) Dihydroli poyl transacetylase -lipoate & CoA

o (12) Dihydroli poyl dehydrogenase - FAD & NAD

Regulation

o Inhibited by: ATP, acetyl-CoA, NADH, fatty acids

o Stimu lated by: AMP, CoA, NAD+, Ca2

+

Citric acid cycle acetyl-CoA 3NADH + 3H+ + 2C0 2 + FADH2 + GT

substrate product notes

1 citrate synthaseacetyl CoA citrate

oxaloacetate + H2O

2 aconitase citrate isocitrate

isocitrate dehyd rogenaseisocitrate a-ketoglutarate

3NAO+ NAOH + H+, CO 2

4 a-ketoglutarate dehydrogenase complexa-ketoglutarate succinyl-CoA heme

NAO+ NAOH + H+, CO 2



5 succinyl-CoA synthetasesuccinyl-CoA succinate

6 succinate dehydrogenase

7 fumarase

GOP

succinate

Q(FAO)

GTP

fumarate

fumarate malate f- urea cycle

~ 8 ~ ~ ____ ~ a ~ l a ~ t e ~ d ~ e h ~ y ~ d ~ r ~ ~ g e ~ n ~ a ~ s ~ e ______ L m r a ~ l ~ a t ~ e ~ N ~ ~ f V ~ __ o ~ x ~ a ~ l o ~ a ~ c ~ e t ~ a ~ t e ~ ~ N ~ A ~ O ~ ~ ~ _________

c reactions - cofactor - thiamine pyrophosphate

replenish CCA intermediates

pyruvate carboxylase- pyruvate + CO2 o ~ a l o a c e t a t eo biotin - prosthetic group - COf-carrier

o important to gluconeogenesis CCA

o stim acetyl-CoA

ketone bodies - accumulate when gluconeogenesis in liver

depletes oxaloacetate (acetyl-CoA oxidized into ketones)

o synth in liver mitochondria

o acetone, acetoacetate, D-6-hydroxybutyrate (feeds brain)

o converted back to acetyl-CoA in brain, muscles for fuel

of Citric Acid Cycle

Enzyme Inhibits Stimulates

ATP, acetyl-CoA, AMP, CoA,

NADH, fatty acids NAD+, ci+

citrate synthaseATP, citrate, NADH,

ADPsuccinyl-CoA

dehydrogenase ATP ADP,ci+

a-ketogluta rateNADH, succinyl-CoA ci+

e cycle (modified CCA in plants, microorganisms, etc.)

allows synth of sugar from fat (acetyl groups to succinate)

step 1-3, ident ical to CCA (acetate -> citrate -> isocitrate)

o bypasses steps in which carbon is lost as COz

isocitrate dehydrogenase

o pp'd > glyoxylate cycle

o depp'd -> citrate acid cyclestep 4 isocitrate -(isocitrate I y a s e ~ succinate + glyoxylate

o succinate CCA

o glyoxylate + acetyl CoA - malate y n t h a s e ~ malate

CCA / gluconeogenesis

used to make nucleotides coenzymes

o RNA, DNA, ATP, NADH, FADH 2 CoAl

controlled by NADP+ availability

NADPH produced used in anabolic reactions (fatty acid synth)

+ 2 NADP+ + H20 -> ribose 5-phosphate + CO 2 + NADPH + 2H+

phase

Reactants Product I Enzyme

IG6P + NADP+ 6-phosphoglucon I glucose 6-phosphateI • dehydrogenase

+ H20 6 P h O ~ 6-phosphoglucolactonase

6PG + NADP+ ribulose 5-p 6-phosphogluconate

NADPH +CO 2 dehydrogenase

R5P ribose 5-phosphate I phosphopentose isomerase

I Acceptor Regeneration phase

sugar phosphates 56-carbon phosphates

NU LEOTIDES NITROGEN Y LE

Nucleotide Precursors

- AAs, ribose 5-phosphate, CO 2, NH3

- N for purines from glutamine, glycine, aspartate

- aspartate precursor of pyrimidines

ribonucleotide reductase - ribose to deoxyribose NDPs

adenylate kinase - 2 ADP _ M g 2 + ) ~ ATP + AMP

de novo pathway

- pyrimidine bases assembled prior to attachment to ribose

o carbamoyl phosphate + aspartate + ribose 5-phosphate

o 5-phosphoribosyl-l-pyrophosphate PRPP)

• initial acceptor of pyrimidine ring framework

o orotate - a pyrimidine ring framework + riboseo UMP UTP CTP (cytidylate synthetase)

o dUMP + N5,N

1o-methylene-THF ~ dTMP

- purine bases assembled on ribose

o ring built from 5-phosphoribosylamine (committed step

o A G derived from inosine monophosphate IMP)

o coenzyme: tetrahydrofolate

o IMP adenylosuccinate AMP

o IMP xanthylate (XMP) GMP

salvage pathway

recycled base + 5-phosphoribosyl-1-pyrophosphate PRPP) NM

Nitrogen cycle

- bacterial fixation - Nz NH3

o 16 ATP per N2 fixed (2 ATP per electron pair added)

o symbiotic bacteria in roots of leguminous plants

o access to large energy reservoir of plant

- soil bacteria - NH3 N0 3-

o plants - N03- NH3

- all organisms - NH3 AAs nucleotides

- denitrifying soil bacteria - N0 3- Nz

Nitrogen transport

- glutamate - intracellular ammonia carrier

- glutamine alanine - extracellular ammonia carriero excess transported to liver Urea cycle)

Transaminases

- glutamine synthetase

o main regulatory enzyme in nitrogen metabolism

o glutamate + ATP + NH3 glutamine + ADP + phosphate

- glutaminase - glutamine ~ glutamate + NH/

- glutamate dehydrogenase

o glutamate ~ a-ketoglutarate + NH/

Urea cycle

- birds reptiles excrete uric acid (uricotelic animals)

- takes place in liver mitochondria cytosol



NH4' -7 carbamoyl phosphate -7 urea

1 N from NH/, 1 N from aspartate

Reactants Products Enzyme

2ATP+ carbamoyl phosphate carbamoyl phosphate

HCO g + NH/ 2 ADP + i synthetase I (mito.)

carbamoylornithine

phosphate + citrulline + Pi

ornithinetranscarbamylase (mito.)

citru lline + argininosuccinate argininosuccinate

aspartate + ATP +AMP+PP; synthetase

argininosuccinate arginine + fumarate argininosuccinate lyase

arginine + H2O ornithine + urea arginase

REPLICATION- temperature at which half of DNA helices melt apart

DN absorbs more light than annealed

forms

B form - right-handed, Watson-Crick structure

A form - more compact (wider, shorter) than WC

Z form -left-handed, longer + narrower

tein pairing - DNA that allows for trip lex DNA strands

4 strand binding between guanosine

A - mirroring leads to triple helix

- inverted DNA repeat - form hairpins or cruciform

2-fold symmetry across 2 strands of DNA

can help dislodge/unbind histones

plectonemic interwined in simple and regular way

o extended r i g h t ~ h n d e d coils

solenoidal tight left-handed coiling

# circular DNA strand wound about another

(LK Tw + Wr, Tw = wist #, Tw =writhe number)

-linked DNA strands (like links in a chain)

- Type I breaks one strand, retains energy to reseal (no ATP)

o DNA gyrase negatively supercoils (underwinds) DNA

- Type II - breaks both strands

- DNA is inverted causing change in gene expression

type locus - changeable sequence where mating type stored

ng - fusion of two haploid cells

& cro repressor proteins - repress other's synth

prophage & lytic states

largely consist of short, repeated DNA sequences alpha

satellite DN

over hundreds of thousands of bps

do not seem to contain centromere-specific DNA sequences

defined by protein assembly rather than specific DNA seq

island

CG least frequent dinucleotide

o large % near of promoters

• methylat ion may inhibit expression

CG suppression -loss of Cs due to methyl + deamin (C-7T)

Genome

LINEs - long interspersed nuclear elements (>Skb)

retrotransposons that lack LTRs (long terminal repeat)

SINEs short interspersed nuclear elements «500

Retroviral-Iike elements

DNA-only transposoh 'fossils'

SSRs (single-sequence repeats/highly repet itive sequences)

generally less than 10bp repeated up to a million times

satell ite DNA - unusual base comp causes it to migrate as

satellite bands (separated from rest of DNA)

Transposons (mobile genetic elements)

produce many genetic variants necessary for evolution

Complexity

insertion sequences simple)

o seq required for transposition & transposase genes

complex transposons - contain one or more genes in additio

to those needed for transposition

Types

DNA-only transposons responsible for antibiotic resistance

o Bacteria contain mostly DNA-only (few nonretrov iral

retrotransposons)

o exist only as DNA move by cut-and-paste transposi tion

replicative transposition

retroviral-Iike retrotransposons

o mRNA -7 reverse transcriptase -7 RNA/DNA -7 DNA/DN

-7 integrated by intergrase

o require RNA polymerase & reverse transcriptase

nonretrovira l retrotransposons

o responsible for repeated DNA seq

o require endonuclease + reverse transcriptase complex

movement

o direct transposition - movement from one site to anoth

o replicative transposition - replication into new site

Transposon-related terms

transposase protein that promotes transpositions

intergrase - viral transposase

cointegrate donor & target DNA intermediate during replicative

transposition

Genetic Variation

SNPs (single-nucleotide polymorphisms)

extensively characterized i n t r ~ s p e c i e s gene variations

Genes

haplotype blocks - sets of alleles that are inherited in clusters

complex traits - do not follow simple inheritance (often polygenic

site-directed mutagenesis - intential mutation to determine funct

transgenes - foreign or modified genes

synteny same genes in the same order across species

pseudogene - duplicated gene deactivated by mutations

X-inactivation - occurs after Nl000 cells formed (mosaic)

x-inactivation center - XIST RNA

10% of genes remain active

unique histone composition/modification pattern

Barr body - condensed inactivated x-chromosome

dosage compensation

o inhibition, up regulation, down regulation



heterochromatin - highly condensed, inactive

euchromatin less condensed, active (acetylated histones) ,

o hypersensitive sites

• actively transcribed regions sensitive to DNase I

AT sequences more compressible

position effect - gene silencing of positions win or nearby

heterochromatin

chromatin jiber - 30nm, zigzag model of packing

metacentric - middle centromere

submetacentric - slightly off-center centromere

acrocentric - more off-center

telocentric centromere at or near end

isochromosome -lost arm replaced by duplicate of other

highly conserved, rich in lysine arginine +)

structure: fold - 3 helices + 2 loops

N-term tail covalent modification determines chromatin

o Acetylation/methylation of Iysines

o Phosphorylation of serines

o H4 tai l- important for nucleosome-nucleosome linking

C-term tai l- mono or polyubiquitinated

variant core histones

o expressed at different times during development

o CENP-A - centromere-specific H3 histone

Hl-l inker histone (larger less conserved)

DNA sequences - protect spread of histone code

HS4 sequence - protects l3-globin expression

20 sets of histone genes

synth mainly in Sphase (1't ranscription, -J.,degradation)

HATs histone acetyltransjerases)

o neutralize +) charge, destabilizing chromatin

o (type A - nuclear, type B - cytosolic)

HDACs histone deacetylases)

o compacts chromatin, attracts other proteins

histone chaperones chromatin assembly factors

SW1/SNFNURF

- remodel chromatinSMC proteins structural maintenance of chromosomes

different combinations of tail modification important to

o marking

• replicated chromatin

• DNA damage

o Inactivation

Heterochromatin formation

Hox silencing (developmental gene silencing)

• X chromosome inactivation

Code-reader complex - adapter that interprets histone code

recruits protein complexes

nucleosome

basic unit of chromosomal organization, N200 bps

o linker DNA - up to 80bp

o nuclesome core

histone octamer 2x H2A, H2B, H3, H4)

• dimers H3+H4, H2A+H2B) tetramers (2x H3+H4)

• 147bp DNA (1.65 turns), left-hand coiled

location - depends on seq, presence of other bound protein

nucleosome sliding - movement along DNA catalyzed by

remodeling complexes

polytene chromosome

bands (dark) - inactive

interbands (light) - active

puffs (Balbiani rings) - transcriptionally active DNA

DNA binding motif s

helix-tum-helix

o regulatory proteins -lac trp, CAP

o homeodomain - developmental

zincjingers hold together by zinc

o steroid receptors

leucine zipper moti f 2 alpha helixes - dimer

o transcriptional activators

helix-loop-helix HLH) short a-helix + loop + long a-helix

P-Ioop

Damage/Mutation

Damage

deamination of bases loss of amine)

o C 7 U - 100 per day per cell

loss of bases - bases dissociate from ribose (N-glycosyllinkag

o depurinat ion 5000 per cell per day

o depyrimidation - 500 per cell per day

UV induced pyrimidine dimerso result in deletion or substitution of 1+ base pairs

oxidative damage occurs to double bonds C-C bonds

hydrolytic attack - N-C bonds, phosphate

uncontrolled methylation - nitrogen, flipping out

radiation - breaks purine rings phosphodiester bonds

Tautomeric shifts

cause G-T A-C binding

G T - keto 7 enol

A amino 7 imino

Mutation rate

1 in 10

nucleotides

20,000 DNA alterations per cell per day

Mutations

point (single site} - single nucleotide or very small part

o transition one purine (or pyrimidine) to other

o transversion purine to pyrimidine vice versa

o nonsense mutation - results in premature termination

o missense mutation different translated M

insertion / deletion loss of or gain of nucleotide(s)

o jrameshift insertion/deletion altersing reading frame

inversion - segment is reversed

translocation - segment migrates from one chromo to anoth



germ-line - allows mutation to be passed to progeny

polar- mutation that affects subsequent genes (polycistronic)

Mutations

conditional phenotypic change under cert. conditions (temp)

loss offunction reduced gene act ivity (usually recessive)

o null complete loss of gene activity

gain of unction - increased or inappropriate activity (usually

dominant)

o dominant-negative - mutant interfereswI normal gene

suppressor- counteracts phenotypic effect of prior mutation

o back mutation reversion to previous sequence ' 'o intragenic - with in 1 gene

o extragenic - within 2 genes

cal repair

photolyases - use light energy to reverse dimmers

- depends on methylation of A resides (in GATC seq)

sion repair

excision repair

DNA glycosylase exinuclease)o removes base from DNA lesions (creates AP site)

AP endonuclease

o cuts phosphodiester at AP (apurine/apyrimidine site)

cleaves phosphodiester backbone on both sides of distortion

DNA around site of damage is excised & resynthesized

TFIIH - opens up excised DNA

repair more efficient wIn actively transcribed genes

RNA polymerase stalls on lesions & directs repair machinery

ologous end-joining ends ligated wI loss of 1 nucleotides

requires long homologous segments

exonuclease 5' end, strand invasion, branch migration

genetic exchange between DNA wI extensive homology

occurs wI highest frequency during meiosis prophase I

resolution by crossover or non-crossover (gene conversion)

can repair DNA damage & restart replication forks (primosome)

too much (loss of heterozygosity)

too little (mutations lead to cancer)

- integrates & excises during recombination

can result in inversion, turn ing genes on or off

lliday intermediate - X orm

ladderlike structure blw homo. chromosomes during meiosis

interconnected by transverse filaments TFs)

- individual crossover events blw nonsister chromatids

only visible during diplotene

ensures at least one crossover blw homologs

crossover inhibits formation of others

of Replication)

rich in AT (weaker hydrogen bonding)

binding site for aRC, aRC recruitment proteins

10,000 in humans excess ensures replication pr ior to divisio

o 30,000-250,000 np bIn replication origins in mammals

replication unit - origin cluster activated together (20-80)

o activate in reproducible order

ARSs autonomously replicating sequence)

o Euk. chromosomal origin of replication

ORC Origin Recognition Complex)

remains associated wI origin through cell cycle

pre-RC pre replication complex)

aRC, DNA, Cdc6 & Cdt l helicase loading proteins

form only during Gl phase Cdk activity low)

2x Cdc6 + 2x Cdtl recruit 2x Mcm - helicase 6 subunits)

geminin - binds & inhibits Cdtl

disassembled during 5 phase (1'Cdk, Mcm pp'd starts repl.)

l 5 & M-Cdk & .J APC/C activity blocks pre-RC formation

primosome DnaB helicase + DnaG primase (lagging strand)

replisome - entire replication complex at replication fork

DNA replicationIn prokaryotes:

DnaA binds 4x 9-bp

HU denatures DNA in 3x 13-bp repeats

helicase further unwinds DNA

SSB proteins bind RPA in eukaryotes)

E. coli DNA polymerases

DNA polymerase I

removes RNA primers

Klenow fragment DNA poll wI removed 5'->3' exonuclea

DNA polymerase III

more processive than DNA poll blc of subunits

a subunit - polymerization activitysubunits - DNA clamp, prevents dissociation

E subunit - 3' to 5' exonuclease activity

T subunits links 2 core polymerases & coordinate synthes

Mammalian DNA polymerases

DNA polymerase a - synth primers & start of Okazaki fragments

DNA polymerase - base excision repair

DNA polymerase 6 - primary pol for both strands

DNA polymerase V - mitochondrial DNA

Viral DNA polymerases

DNA viral genomes encode own DNA polymerase

Mitochondrial & plastid DNA - circular, 2 ori, multiple copies (2-1

RN TR NSCRPTIONtranscriptome - all transcribed genes in an organism mRNA + rRN

RNAs

snRNAs (small nuclear) splice pre-mRNA

snoRNAs (small nucleolar) - modify rRNAs

many encoded in introns of ribosomal protein genes

scaRNAs (small cajal) modify snRNAs + snoRNAs

miRNAs & stRNAs (micro & small temporal )

complementary to 3' UTR of mRNAs

block translation & target mRNA for degradation



initiates RISC (RNA-induced silencing complex) formation

As - small interfering compact chromatin & degrade mRNA

- telomere synthesis,x-chromosome inactivation

& ribosomal proteins

200 rRNA gene copies per haploid genome in humans

precursor

~ 1 0 0 methylations (2'-OH positions)

~ 1 0 0 isomerizations (uridine -> pseudouridine)

modified & cleaved by snoRNAs\

also contain genes for DNA primase, RNA polymerase, EFs

r-protein in each operon functions as repressor

o repression if r-proteins exceeds rRNA

RNase 1/1- trims pre-rRNA

heavily modified (over 50 types of modification)

some eukaryotic tRNAs have introns

Structure

o amino acid arm

CCA 3' (added by tRNA nucleotidyl transferase),

• AAattached to either 2 or 3 hydroxyl

• RNase P - cleaves 5 of tRNA

• RNase D - trims 3 of tRNA

o anticodon arm

o D arm & TtjJC arm

- different anti-codon tRNA coding for same AA

synthetases Mg2+ dependent, 2 steps, ATP

- inosine/inosinate in 3 position base-pairs w/ U, C, or A

5' wobble - GUG may initiate translation in prok. ( ~ 1 4 )o UUG & CUG even less frequent at start codon in prok.

moncistronic - code for single polypeptide (most all euks)

polycistronic - code for multiple polypeptides (prok. & viruses)

UTR (untranslated region)

region bIn stop codon & poly-A tail

plays role in mRNA localization & transcrip tion con trols

repressors disrupt comm. bIn tail & cap - slowing translation

carry sites for proteins that accelerate/impede degradation

editing

uracil insertion/deletion

o{gRNA (guide RNA) templates} - frameshifting

A 7 I (inosine) occurs to dsRNA

o ADARs ADR1 liver, ADR2 - bra in, ADR3 - unknown

C-7U

forms RNA-like polymers from RDPs w/o template

Involved in mRNA processing & degradation

Degradation

large protein complex that degrades RNA w/ exonucleases

processing bodies) - cytosolic site of most RNA degradation

RNA Polymerase ,/ Iv.

does not require DNA helicase (ATP)

deacetylates histone tails

limited proofreading (no nuclease activity)

requires template, nucleotides, Mg2+ & Zn2+

pro:

o RNA polymerase holoenzyme

• core enzyme a a ~ w(J subunit/factor

• specificity factor

• binds to promoter

• dissociates from core ns into translation

eu: (3 pols - differential sensitivities to a-amanitin)

o RNA polymerase 1- 5.85, 185, 285 rRNA (45S precursor)

o RNA polymerase II - mRNA

• continues trans, but overshoot lacks cap (degrade

o RNA polymerase 111 tRNA, 55 rRNA, snRNA, other

CTD (C-terminal Domain)

o responsible for initiation & bonding of related complexe

o cap synthesizing complex

• synth 5 cap (5' to 5 7-methylguanosine - protects

eu-mRNA & aids in translation)

o cap binding complex

secures mRNA to RNA polymerase following cappi

Initiation

PIC preinitiation complex) - denatures DNA & helps position RNA

polymerase II

no uniform order of assembly

o (TFIID 7 TFIIB 7 TFIIF 7 TFIIE 7 TFIIH)

consists of 6 general transcription factors

o TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

(1) T F I I D ~ T B P (TATA-binding protein) binds TATA box

(2) TFIIB - binds TBP & DNA

3) TFIIA - stabilizes TFIIB-TBP complex (not necessary)

important for nonconsensus promoters which bind TBP wea

(4) TFIIF + Pol II bind TFIIB-TBP complex

(5) TFIIE & TFIIH bind - create closed complex

Transcription initia tion complex - polymerase + transcription fact

TFlIH - unwinds DNA to start transcription ATP)

o Phosphorylates RNA polymerase CTD (C-terminal doma

o Releases polymerase from promoter

Elongation

TFIIE & TFIIH - released in initial 60-70 nucleotides

Transcriptional elongation factors

suppress pausing, decrease likelihood of dissociationcoordinate activites between protein complexes

involved in posttranscriptional processing

dephosphorylated upon termination

p TEFb positive transcription elongation factor b) pp's CTD

Polyadenylation

upstream AAUAAA site

CstF cleavage stim factor) & CPSF cleavage & polyadenylat

specificity factor) transferred from tail to 3' end

PAP polyadenylate polymerase) - adds ~ 2 0 0 A (from ATP)

o slow addition at first followed by rapid addition of 200-

tail acts as timer (mRNA half-life)

o short for bacteria (seconds)



o varies for euks - long for B-globin (10h), short for growth

factors & regulatory proteins

tail can be extended, extending mRNA life

Poly-A binding proteins - determine final length, help direct

translation

ional controls ( ~ 7 5 of human genes)

transcription attenuation premature termination

o used by many AA synthesis operons

riboswitch secondary structure of mRNA that turns on/off

additional transcription

alternative RNA splicing

quality control

cytosol

5'cap & poly-A tail recognition

EJ (exon junction complex) - stimulates translation

Nonsense-mediated mRNA decay

o stop codon in wrong place causes degradation

ns - exon size much more uniform than intron size ~ 1 5 0 )Introns

Composition: snRNP (RNA + protein) - snRNAsLocation: bound to pp'd tail of RNA polymerase II

SR proteins - mark junctions - recruit snRNPs

U1- 5' splice junction

BBP & U2AF - branch point, mark upstream boundary

U2 - branch point

U4/U6*U5 - triple snRNP - splices

pe splicesome +10 to all U s, recognizes diff nucleotide seq.

splicing RNA - rare - phage, mitochondria, chloroplast genes

Group 1- G attacks, no lariat (guanosine cofactor)

Group II - A attacks, lariat

(rare) - mRNA from diff mRNA spliced together

single 5' exon spliced onto different 3' exons

search for identical site for intron insertion

homing endonuclease copies intron into new homo. gene

retrohoming DNA reverse spliced - spliced intron inserted

by endonuclease reverse transcrip tase

egulation

control region gene promoter & regulatory sequences

polycistronic region translated together from single promotergene + promoter, operator, enhancer, etc

network of operons wi common regulator

HREs (hormone response elements)

o reg. DNA seq binds hormone- receptor complex

operator - bound by repressors & activators

TATA box (Hogness) - 25 bp (bound by TBP)

Pribnow box - -lD bp (binds RNA polymerase subunit)

additional reg. seq. in higher euks bound by activators

can enhance transcript ion of upstream & downstream genes

UASs (upstream activator sequences) in yeasts

UP element (upstream promote r) -40-60 bp

CAAT box - 75-80 bp

initiator start of transcription - +1 bp

terminator sequence that ends transcription

Trans-elements

DNA-binding transactivators bind enhancers/UASs

effector regulates repressor binding

inducers prevents binding of repressor protein to DNA

gratuitous inducer inducer cannot be metabolized

basal transcription factors required at every RNA pol II promote

coactivators/ corepressors do not themselves bind on DNA

mediator mediates activator & transcription machinery

.RNA sequences

Kozak sequence translational start site for ribosome

leaCler - 5 ' end of mRNA preceding initiation codon of first gene

attenuator -lies within leader responsible for attentuation

start codon AUG

stop codons GAA, UGA, UAG

Specific examples

catabolite activator protein (CAP) regs certain prokaryotic genes

complexes wi cAMP, binds promoters

lac operon - transcribedwi

low glucose conc. ([cAMP] high) & higlactose conc. (Lac repressor unbound)

CRE & CRE cAMP response element-binding)

TR NSL TION

Ribosome

small subunit - framework for matching

o pro: 165 rRNA from 305 pre-rRNA

o eu: 185 rRNA from 455 pre-rRNA

large subunit

o pro: 235 & 55 rRNAs from 305 pre-rRNA

305 contains tRNA as intronso eu: 5.85 & 285 rRNAs from 455 pre-rRNA

o catalyzes peptide bond - peptidyl transferase

o Sites:

E - ex it

P - peptidyl-tRNA

A - aminoacyl-tRNA

polyribosomes/polysomes multiple ribosomes translationa

complexed on ER surface

Initiation

Initiation in pro

Translations begins while transcription is still in progress

Initiation complex

o 305, mRNA, fMet_tRNAfMet

, (IF-1, IF-2, IF-3), GTP, Mg2+

305 binds IF-1 + IF-3

o IF-3 prevent premature 305 + 50S binding

mRNA binds 30S-IF-1-IF-3 complex (SD seq pairs wi 165 rRN

fMet_tRNAfMet + IF2-GTP bind AUG

50S subunit binds - GTP hydrolyzed, initiation factors depar

Initiation in Eus

elFs (eukaryotic initiati on factors)

5' & 3' ends tied together - PAB (Poly(A) binding protein)

elF4F complex (eIF4E, elF4G, eIF4A) bindsto 5' cap

o [eIF4A- RNA helicase]

elF4G - binds elF4E & PAB



elF4F complex - associates w/ elF3 & 405 ribosomal subunit

elongation factors EFs) increase speed & accuracy

binding aminoacyl tRNA (aa-tRNA+EF-Tu-GTP)

o EF-Tu - mediates aminoacyl tRNA entry to ribosome

peptide bond formation (235 rRNA - peptidyl transferase)

translocation (EF-G(translocasel + GTPl [EF-G catalyzes

translocation of tRNA & mRNAJ

o large subunit translocation, then small subunit

translocation

Ts - serves as GEF guanine nucleotide exchange factor) for EF Tu

- initiation on free ribosomes

- binding of 5RP (sig rec. particle) to polypeptide (GTP-used)

- binding of 5RP, polypeptide, & ribosome to 5RP receptor

- dissociation of 5RP from its receptor

- translocation across membrane of ER

- cleavage of signal sequence

translocon -takes signal seq across ER memb

- termination codon in the A site

anslational modifications

In Lumen of ER and/or Golgi

N-term & C-term modifications removed or modified

Loss of signal sequence

proteolytic processing - inactive to active

Phosphorylation 5er/Thr/Tyr

Carboxylation Glu

Glycosylation usually occurs in Golgi

o Asn - N-Iinked oligosaccharides

o 5er/Thr - O-linked oligosaccharides

o isoprenyl groups - Cys - thioether bond

(helps anchor protein in mem.)

o prosthetic groups - biotin, heme

disulfide cross-links

quaternary structure - complexing of subunits

phosphorylation of initiation factors

RNAi - miRNAs, stRNAs bind mRNA (to 3' UTR)

o prevent translation initiation

disrupt elF4E & elF4G interaction

UAA, UAG, UGA

causes binding of water molecule instead of amino acid

interference

dsRNA inhibits genes w/ complementary sequence

can lead to heterchromatin formation

7 2'-S'oligo(A)synthetase 7 RNase L (destroys aU RNA)

7 Protein kinase R 7 apoptosis

high energy phosphate bonds split for each new peptide bond

2 consumed in charging of tRNA w/ AA ATP -> AMP)

2 during synthesis in ribosome

extra energy consumed w/ mistaken connection

Regulation & modification

Translational repressors bind 3' UTR (untranslated region) of mRN

block interaction w/ initiation factors or ribosome

prevent or slow translation

Reading Frame

ORF open-reading frame) lacking termination win codons

translation recoding

proceeding seq. causes codon to be translated differently

translational rameshifting

shift of reading frame as ribosome is translating mRNA

.leaky scanning - ignoring of AUG codon, produces differen t prote

" different or no N-terminal signal sequence

IRES internal ribosome entry site) - bypass need for 5' cap & elF4E

CELLUL R COMPONENTSNucleus

Subnuclear structures

Cajal bodies & GEMS (Gemini of Cajal bodies) - srioRNAs &

snRNAs modify al lItil8g l11fjli i . NS

IGCs (lnterchromatin granule clusters) - stockpiled mature

snRNPs

nucleolus - site ofrRNA transcription

o NOR (nucleolar organizing region) - region containing 4

pre-rRNA genes around which nucleolus forms

Nuclear lamina

protein meshwork, provides support for nuclear membran

disassembles through phosphorylation by Cdk

Nuclear pore complex NPCs)

aqueous, permeable to small molecules «50,000 daltons)

octagonal w/ long filaments

Karyopherins (nuclear transport receptors)o Export

• 5' cap first CBC (cap-binding complex)

• sheds some proteins (nuclear-restricted proteins)

• e e & nuclear export receptor h . ~ c in cytosQL.;

• Ran-GEF in nucleus causes R a n - G t ~ : - - > o o R a i l - G D J yo Import erf .) ({ .

• Ran-GAP in cytosol causes R a f l ' 6 ~ ~• importin bound (via FG-repeats)

• Nuclear import receptors - bind NPC signal

Endoplasmic Reticulum

lumen/cisternal space

cytochrome P450 system

o oxidative metabolism in ER o iver & small intestine

o drug metabolism & detoxification of foreign substanc

sequester ci+ - specific regions of ER specialized for stora

lipid synth - most lipids synth in cytosolic leaf of ER

membrane

o cholesterol, ceramide & fatty acid

protein modification

o glycosylation

o proetolyt ic processing - in ER & post-Golgi

Transport into ER

Most proteins imported during translation



Signal-recognition particle SRP) - cycles in ER cytosol

Sec61 complex - tetra mer, 1 pore + bind ribosome, proteins

start transfer peptide - binds w SRP - starts translocation

o internal start transfer signal can serve as anchor

stop transfer peptide - anchors protein in membrane

o multipass transmem. proteins - internal start,

alternating stop start seq.

ER resident proteins

o ER retention signal - K[)El (lys-Asp-Glu-leu)

o most complex - too large for most t ransport vesicle's

post-translation translocation requires motor protein

o BiP - pulls in protein

cis - ER side, trans - plasma membrane side

Anterograde transport through the Golgi apparatus occurs

via cisternal progression

catalase urate oxidase (crystallized core)

o c t l se - hydrogen peroxide -> w ter + oxygen

o use O2 to remove H from organic substrates 13

oxidation)

Import

o peroxins - import proteins into peroxisomes

o C-term peroxisome import seq. - Serine-lysine- leucine

o Unknown N-term sequence

osomes - acid hydrolases

hydrolyzes cholesterylesters

o releases cholesterol fatty acids into cytosol

mannose 6-phosphate M6P) - targets proteins for

Iysosomes

osomes - acidic H+ATPase)

early, late recycling - recycles stores membrane proteins

receptors, etc

s (MVBs) - endosome w invaginations

sorting into internal vesicles regulated by ubiquitin

- cytosol

- single-membrane of vacuole

- photosynthetic membrane invaginations

- invagination to which DNA is attached

germ cells PGCs)

cells singled out early on gamete progenitors

development

(sex-determining region of Y) - directs testis development

ls - main suppor t cells in testis

greater quality control than egg development

contribute more mutations (larger number of mitotic divisions)

acrosomal vesicle - secretory vesicle at head of sperm

syncytium - mUltiple nuclei w shared cytoplasm

spermatogonium - rep.)-7 prim. spermatocyte - mei-l)-7 sec.

spermatocyte -(mei -II)- 7 spermatid - spermatozoa (mature sperm

oogonium - rep.)-7 prim. oocyte -(meiOSiS 1)-7 sec. oocyte - mei-

11)-7 oovid - ovum (mature egg)

egg cells

main source of aneuploidy

parthenogenetic - eggs activated in absence of sperm

nurse cells - feed mRNA into oocyte

Fertilization - usually egg is a secondary oocyte

1) capacitation - sperm modif. by female reproductive trac

o changes in glycoproteins, lipids, ion channels RP

o unmasks cell-surface receptors that help sperm bind

o greatly increases flagellum motility

. (2) reach site of fertilization in oviduct

(3) migrate through granulosa ~ l l s surrounding egg

4) bind to cross zona pellucid

(5) bind to fuse w plasma membrane

o fusion activates egg by increasing Ca2+ in cytosol

o sperm donates centrioles (lacking in unfert ilized egg)

polyspermy - more than one sperm fuses

polyspermy blocks

fusion of first sperm causes change in egg plasma membrane

cortical reaction - enzymes change structure of zona pellucid

o second polar body is extruded

aneuploid - cells w abnormal chromosome

euploid - cells w normal chromosome

in vitro fertili zation lVF) - aids infertile women

intracytoplasmic sperm injection lC51) - aids infertile men

Inheritence

genomic imprinting - expression of maternal or paternal gene onl

epigenetic inheritance - inherited protein structure superimposed

on genetic (DNA-based) inheritance

m tern l effect - accumulated mRNA of mother's genes that

dictates early development

OTHER ELL TYPES

Stem Cells

Depend on contact signals from strom l cells

totipotent - any cell type, including extraembryonic

pluripotent - any cell type, but extraembryonic cells (placen

immort l strand hypothesis - stem cells maintain same DNA

transit amplifying cells - divide frequently, leave basal layer

terminal cell differentiation - final specialized characteristics

cellysually permanently stops dividing

apical meristems - undifferentiated pluripotent growing tip in pla

Epithelial cells

keratinocytes - epithelial cells w excessive keratin intermediate

filament synthesis which give epidermis its toughness



Cells

- construct bone

osteocyte imprisoned in hard matrix

- remove bone (control led by osteoblast signals)

cells

abundant smooth ER (principle site of lipoprotein production)

differentiated cells, no apparent stem cells .

interface between digestive tract and blood

major function - maintain blood glucose level G6Pase) ,

- air-filled sacs of lungs

Type most of wall, thin flat to allow gas exchange

Type I interspersed among Type I, plump, secrete surfactant

antimicrobial peptides that kill p a t h o g ~ n s in mucus layer

most abundant proteins in neutrophils

short (12-50 AAs) positively charged

et cells, ciliated cel/s, macrophages - keep airways cleangoblet cells - secrete mucus

tive cells brush-border cells or enterocytes)

microvilli increase nutrient uptake secrete digestion enzymes

cells

secrete serotonin peptide hormones that act on gut wall

cells - innate immune cells in small intestine

similar to neutrophils, secrete cryptdins (defensin family)

cell niche crypt in gut lining that protects stem cells

acids enzymes

- stims secretion of HCI into stomach, optimal for pepsin

acid - low pH acts as a denaturing agent

- (chief cells) cleaves phe,leu not next to proline}

- cleaves tyr , phe, leu, lie, val, trp, his at high pH

- (duodenum) cleaves lysine arginine not next to proline)

buffering

CO2 + H20 -> H+ + HC03-

cells

l cells - form linings of blood vessels lymphatics

- cell fragments derived from megakaryocytes

(rouget cell, mural cell) .- blood vessel suppor t cells

differentiate into fibroblast, smooth muscle, or macrophage

proteins

n - carries iron in the blood

albumin carries fat ty acids to heart skeletal muscle

lood cell promoting hormones

CSFs)

support hemopoiesis (formation of blood cellular components)

stims

o erythropoietin stims production of erythrocytes

o interleukin-3 lL3) - promotes survival proliferation of

earlier erythroid progenitor cells

lood cell lineage

BFU-E

myeloid stem cell

pluripotent hematopoetic stem cell (hemocytoblasts)

Reticulocyte - immature erythrocyte

Erythrocyte - red blood cell

Hemoglobin

heme - porphyrin ring, synth'd from s J c c i n y / ~ C D AR state - relaxed (higher O2 affinity) / T state - tense (lower

affinity)

O2 bindingo moves heme iron closer to porphryn ring plane

o prevents binding of 2,3-bisphosphoglycerate 2,3-BPG)

lowers O2 affinity

o converts T to R state

H+ CO2 binding

o binding inversely related to O2 stabilizes T form Bohr

effect)

o H+ - binds to several places.

o CO2 - binds a-amino at N-term of each subunit

o transported from cells to lungs kidneys

fetal hemoglobin - higher O2 affinity than mother's adult hemoglo(due to decreased binding of 2,3-BPG)

Hemoglobin S - abnormal form responsible for sickle cell anemia

Mutant ~ s u b u n i t - (negative AA -> hydrophobic AA)

forms fibrous aggregates when O2 released

Oxygen binding models

MWC model/concerted model - all subunits undergo

transformation simultaneously

Sequential model binding of one ligand makes binding of adjace

ligands more likely

Hill plot -log[6/1- 6] v 10g[LJ

Hill coefficient - slope of Hill plot - a measure of cooperativeinteraction between protein subunits

HORMONESpeptides, amine, eicosanoid hormones act at cell surface

steroid, vitamin 0, retinoid, thyroid horomes act on nuclear

receptors

Peptide hormones

leptin

produced by adipose tissue

regulates energy uptake expenditure

acts on arcuate nucleous of hypothalamus - inhibits eating

sympathetic nervous system - uncouples mit ox ph ->

thermogenesis

functions thru Jak/STAT

ghrelin - produced in stomach - producesfeeling of hunger

adiponectin stim fatty acid uptake / oxidation, inhibits fatty acid

synthesis

functions thru AMPK

atriopeptin - atrial natriu retic peptide/ factor AN P AN F)

hormone secreted atria to lower blood pressure

o reduces water , Na+, adipose loads in blood

vasopressin kidneys reabsorb water



d hormones

- kidneys retain Na+& water

fatty acids

mammals cannot introduce more than 1 double bond into fatfy

acids

obtained from plants

linoleate C-18 cis-t,9, t,12}

a-linolenate (C-18 cis-t,9, t,12,t,lS)

-7 arachidonate (20-C cis-t,s, t,8,t,11,t,14} -7 eicosanoids

glandins, thromboxanes, l e u k o t r i ~ n e s . potent signal

Prostoglandins - regulate synthesis of cAMP (mediate pain,

fever, and inflammation}

Thromboxanes mediates blood Clotting (produced by

platelets)

Leukotrienes powerful signal in aJlergicreactions

- regulate growth/diffe rentiat ion in plants

Plant hormones (growth regulators)

Ethylene - gas molecule

o Fruit ripening, leaf abscission, plant senescence

o Stress signal wounding, infection, flooding

o Stem thickening, shields tip of root, changes growth

direction /ltriple response

auxin - helps plants grow toward light, roots down

- precursors of skeletal muscle fibers

- fuse to repair damaged muscle or allowfor muscle

generally survives for entire animal life time (individualnuclei can be added or lost)

sarcoplasmic reticulum - abundant, modified smooth ER

found in muscle

fast-twitch muscle - fewer mitochondria & less blood supply

than slow-twitch muscle, quicker to fatigue

phosphocreatine / creatine phospha te / phosphagens -

reservoir of high-energy phosphates

myostatin - secreted to limit own growth

nitric oxide relaxes smooth muscle (thru guanylyl cyclase

-7 cGMP -7 protein kinase G}

basic contractile element

sacromere contractile unit

A band- thick filament

I band - only thin filament

M-line - bisects A band (thick filament} where myosin heads

switch polarity (para myosin, C-protein, M-protein)

Z-disk - CapZ & a-actinin - crosslinks & caps +} ends of actin,

bisects I band (desmin}

tropomodu lin - stabilizes minus end of actin

molecular rulers

o nebulin - template protein along length of actin

o titin - links myosin to CapZ

e cycle

1) ATP binds myosin, actin unbinds

(2} ATP hydrolyzed, conformational change & weak binding

F-actin subunit

(3) Pi released, myosin bond strengthened

4} pi release triggers power strokelJ w/ ADP release

Contraction Regulation

tropomyosin- binds actin, blocking myosinattachhlent

troponin T,I,C}

o Toponin 1-T pulls tropomyosin out of normal

binding groove - preventing myosin binding

o Troponin C-binds 1-4 Ca2+causing I to release

actin exposing myosin-binding sites

Neuromuscular transmission

1 nerve impulse causes uptake of Ca2+ (v-gated Ca

2+ chan}

which stim release of acetylcholine

2. acetylcholine binds to nicotinic acetylcholine receptor, in

of Na+ depolarizes muscle

. 3. v-gated Na+ channel lets more Na + in

4. depolarization activates v-gatedCa2+ channels in transve

tubules

5. causes ci gated Ca2+ release channels Ryanodine

receptors) in SR to open - myofibrils in muscle cell contra

Fermentation (regenerates NAD+)

pyruvate -(NADH-NAD+}-7 lactate

pyruvate -> C02 + acetaldehyde -(NADH-NAD+}-7 ethanol

pyruvate -(lactate dehydrogenase}-7 lactate + NAD+

pyruvate -(pyruvate decarboxylase}-7 acetaldehyde

alcohol dehydrogenase-> ethanol + NAD+

o generates molecule of CO 2 from carboxyl

group of pyruvate (carbons 3 & 4 of glucose

acetaldehyde - builds up in liver from ethanol

Cori cycle

when muscles lack sufficient oxygen, pyruvate reduced to

lactatelactate exported to liver

liver converts to pyruvate, then glucose & exported

Glucose-Alanine Cycle

in muscles: pyruvate + ammonia -7 alanine

alaninine transported to liver & converted to glucose & urea

alanine + a-ketoglutarate -7 pyruvate + glutamate -7 glucos

NERVOUS ELLS

neuroblasts - nerve-cell precursors

neurons

- use only glucose & l3-hydroxybutyrate for energy 13

hydroxybutyrate converted to acetyl-CoA via£E1\in brain

-most ATP used for transport of Na+ & K , and maintenance o

potential

glial cells - specialized supporting cells that form myelin

Schwanncells peripheral nerves

Oligodendrites ..:..central nerves

Neuron Ion channels

voltage-gated Na channels responsible for AP (present at Node

of Ranvier along axons)



K channels - reduce AP firing at levels just

that needed for stimulation

results in firing rate that is proportional to strength of

depolarizing stimulus over a broad range

-activated ( channel - allows for adaptation to signal

AMPA receptors - carries most of depolarizing current

responsible for excitatory PSPs (post synaptic potentials)

NMDA receptors - double-gated -glutamate must be bound

membrane must be strongly depolarized

o releases Mg2+

o Ml+ blocks resting chanhel, Ca2

+ nLong-term potential LTP) high activation Ca

2+ evels

activate protein kinases

Long-term depression LTD) modest activation Ca2+

ion concentrations

~ N a + low inside/high outside

- K+ low outside/high inside

- Ca2+ low inside/high outside

- Cl- low inside/high outside

Growth Guidanceneurotrophic factors released by targets guide nerve growth

cone

Eph to Ephrin, Ephrin to Eph, Semaphorin to Plexin

TR NSPORT

Highly Soluble

hydrophobic molecules 02 CO2 N2 steroids, hormones)

uncharged polar molecules H 20, urea, glycerol)

Low to no solubility

large uncharged polar moleculesions

Composition

- phospholipids asymmetrically distributed between layers

o helps w/ signaling, binding, living/dead discernment

- ffippase (p-type pump) - flip-flop diffusion

o moves phosphatidylserine

phosphatidylethanolanine to cytosolic leaflet

- l ipid rafts - high conc. of sphingolipids, cholesterol proteins

Pinocytosis - endocytosis of small parts of membrane forrecycling

Caveolae - caveolins - also capable of endocytosis

Transport

vesicles

Contain

Cargo proteins

Cargo protein receptors

Coat-recruitment GTPases

o Arf proteins - clathrin COPI assembly in Golgi

o SarI proteins COPli assembly in ER

Coat

COPI-coated - from Golgi

CGPII-coated from ER

clathrin-coated -Golgi <-> plasmal membrane <:->

endosomes

o coated pits - endocytosis

PIPs (phosphoinositrides) mark organelles membrane domain

dynamin - forms spiral around neck of bud - helps break.off buds

Rab proteins - guide vesicle targeting, on transport vesicles targ

membraneRab effectors

o membrane tethering - tether first attaches to Rab,

directing vesicle

o motor protein - propel vesicle along actin filament

SNAREs - recognizes proteins for targeting to membs/organelles

v-SNAREs - vesicle

t-SNAREs - target

NSF N - e t h y l m a l e i m i d e ~ s e n s j t i v e factor) separates SNAR

for reuse

secretory vesicles - concentrate cargo, activate by proteolytic

cleavage

synaptic vesicles - partially fused and primed for excretion, locally

recycled and reused

synaptotagmin - Ca2+ sensor, promotes rapid fusion of

docked synaptic vesicles w/ presynaptic membrane

ATP driven pumps

Hype - turb ine like, powered by electrochemical gradien

P-type phosphyrylate themselves during pumping

(Na+,K+,H+,Ca2+ gradients)

o Ca2+ pump Ca2+ ATPase) - sarcoplasmic reticulum

pumps Ca2+ into SR

o Na+-K+ pump (Na+ pump/Na+-K+ ATPase) -ATP driveantiport Na+ out, K+ in

V-type (vacuolar) - proton pumps that acidify membrane

bounded organelles (vacuoles, Iysosomes, endosomes)

ABC transporters-small molecules - ATP-binding cassett

o Mult idrug resistance MDR) protein - pumps

hydrophobic drugs out of cytosol

Overexpression gives cancer cells ability

pump out multip le drugs

Ion channels Na+,K+,Ca2+,Cr)

voltage-gated

o Na+ - skeletal muscle + nerve Na+ down its gradient,

create Action Potential

o K (delayed K channels) K out (down gradient)

returns nerve to negative potentia l

mechanically-gated

ligand-gated

o transmitter-gated - convert extracellular chemical

signals to electric

• excitatory neurotransmitters - open cation

channels, influx of Na+

• inhibitory neurotransrnitters - open CJ- or K+

• neurotransmitters are not exclusively inhibi tory

excitatory



primarily excitatory -acetylcholine, glutamate,

serotonin

• inhibitory GABA (y-aminobutyr ic acid), glycine

o ion-gated, nucleotide-gated

& Transporters

tose transporter / permease proton-driven co-transporter

exchanger (antiport) - H+ out, Na+ in

+-driven Ci HC0 3- exchanger - Na+ & HC03- in, cr & H+ out

3- exchanger - HC03- out facilitates ql ick

of CO 2 in red blood cells ,

+-K- pump - Na + n, K out

leak channels - open even when unstimulated/resting

major-facilitator superfamily) - transport small solutes in

to ion gradients

bind ions for transport across membranes

- H20 pore, high cone. in epithelial cells of kidney

channel-coupled transmitter-gated ion channels ionotrop ic

- GTP-binding protein modulates interaction bin

& target

Most cell-surface receptors in animals are GPCRs

yme-coupled - function as enzymes or associate w/ enzymes

ar receptors - receptors w/ unknown ligands

cyclic AMP, az+, diacylglycerol

(GPCRs) largest family of receptors

Stimulatory G-protein (G,l activates adenylyl cyclase

Inhibi tory G-protein (G;) inhibits adenylyl cyclase +

regulates ion channels

t subunit - binds GDP & GTP

GRKs G-protein receptor kinases) - desensitize/inhibit

GPCR

Examples

rhodopsin receptor

6-adrenergic receptor

o ~ A R K - ~ - a d r e n e r g i c receptor kinase

• pps ~ - a d r e n e r g i c~ - a r r e s t i n ~ a r r ) binds to pp, triggers receptor

sequestrationacetylcholine receptor

. 0 muscarinic acetylcholine receptor (in heart muscle)

opening of K channels

Pathways

Gs adenylyl cyclase cAM PKA CREB + CREBB

DNA-binding

G inhibits adenylyl cyclase & ion channels

Gq 7 phospholipase C ~ { P L C ~ ~ IP3 inositoll;4,S

trisphosphate) + diacylglycerol (DAG) fcleaved from PIPzl

o IP3 7 IPrgated Ca2+ elease channels (ER) ~ C a z + +

calmodulin CaM kinase CREB + CREBB DNA

binding

o DAG arachidonic acid eicosanoids

prostaglandins (pain & inflammatory response)

Enzyme-coupled receptors

Most planet receptors are enzyme-coupled

receptor tyrosine kinases (RTKs) - growth factors

o most enzyme-coupled in animals are RTKs (rare in

plants)

o ephrins - cell surface bound extracellular signals

(bidirectional signaling)

o Eph receptors - cross-phosphorylation - creates dock

sites for signaling proteins (phospholipase C-y)

o Src & PI 3-kinase (phosphoinositide 3-kinase) - pps PH

(pleckstrin homology) where signaling complexes

assemble

o SH2 & SH3 - phosphotyrosine-binding (PTB) domains

tyrosine kinase-associated receptors depend on tyrosine

phosphorylation but lack kinase domain

o antigen & interleukins receptors in lymphocytes

receptor serine/threonine kinases

o most common receptor in plants - LRR (leUcine-rich

repeat receptor kinases)

o te trameric receptor complex of dimer'd type I & type

histidine kinase-associated receptorso two-component signaling pathway

o chemotaxis receptors - methylation responsible for

adaptation

receptor guanylyl cyclases

receptorlike tyrosine phosphatases

Desensitization

receptor sequestration - receptors removed from PM to

endosome(s)

receptor down-regulation - receptor destruction in

Iysosome(s)

receptor inactivation cytosolic domain is blocked

signaling protein inactivation intracellular signaling prote

is inactivated

production of inhibitory protein - pathway produces prot

that inhibits future signal

TR NDUCTION P THW YS

SMAD pathway

inhibits excess cell proliferat ion

T G F ~ (transforming growth factor

T G F ~ T G F ~ receptor II + G F ~ receptor I Smad3 + Smad4

DNA-bindingT G F ~ signal c a u s e s T G F ~ receptor II to pp T G F ~ receptor f

T G F ~ receptor I pp's Smad3

pp'd Smad3 binds Smad4 & is imported into nucleus

Inhibitors I-Smad, Ski, SnoN

Wnt signaling pathway

Wnt secreted signal proteins ( local mediator/morphoge

Frizzled cell-surface receptors

Disheveled scaffold protein

~ - c a t e n i n multifunctional protein

Wnt LRP Frizzled Disheveled -I destruction complex

catenin -I Groucho DNA-binding

lRP & Frizzled binding ofWnt activates Disheveled



Disheveled inhibits destruction complex GSK-3)

~ 7 c a t e n i n inhibits Groucho, allowing LEF binding of DNA

signaling pathway

cytokine receptors - dimers or trimers

JAKs - Janus kinases

STATs - gene regulatory proteins

JAK -7 STAT-binding -7 pSTAT-7

-7 DNA-binding

cytokine brings JAKs together - pps each other, pp cytokine

receptor

STAT docks to pp'd cytokine receptor & pp'd by JAKspp'd STATs dimerize & enter nucleus

pathway

- RTK (receptor tyrosine kinase)

o Insulin receptor

o EGF receptor (epidermal growth factor)

- Ras - GTPase, acts as signaling hub

MAPK - mitogen-activated protein kinase

RTK -7 Grb2 -7 RasGEF SOS) -7 MAPKKK (Raf) -7 MAPKK {Mek -7

(MAPK (Erk) -7 gene regulatory proteins or protein kinases

signaling pathway

conserved developmental pathway

Delta signal- tells adjacent cells not to be neural

lateral inhibition neurons inhibit growth of

additional/redundant neurons

Delta -7 Notch receptor -7 TACE & Presenilin 1 -7 Notch segment

-7 D N ~ b i n d i n g

SIGN LING

of rhodopsin receptor

rhodopsino chromophore i i-cis retinal-light-absorbing

pigment

o opsin - protein portion

ll-cis-retinal is converted to all-trans-retinal

Ught -7 rhodopsin -7 Gt (transducin) 7 cGMP

p h o s p h o d i ~ e ~ i ' t S , , ~ , , ? - . J , . c G t \ I 1 P -7cGMP-gate ion channels close

-7AP

ylate cyclase converts GTP to cGMP

binds phosphorylated rhodopsin, inhibiting it

ins - 109

rod cells & 3 x 106

cone cells

& red cones distinguished environment of not

GPCR) - Golf - cAMP - cyclic-AMP-gated cation

Na+ influx

pain receptors that respond to heat, acidity,

of ions thought responsible for taste sensations

gustducin G protein associated w/ basic taste

Sound

ability to detect range of frequencies due to distinctions in basilar

membrane

PLASTIDS PHOTOSYNTHESIS

Proplastids - no internal membranes or chlorophyll

chloroplast photosynthetic organelle

chromoplast pigment organelleleucoplast storage plastid

o amyloplast stores starch (amylopectin) granule

Chloroplast

thought to have originated from cyanobacterium

thylakoid membrane -light absorption & electron transp

stroma chloroplast matrix

granum a)- stack(s) of thylakoids

make most of own lipids & some amino acids

mitochondria & chloroplasts are maternally inherited in

2/3rds of higher plants

o in some plants, inheritance is biparental

Neither ATP nor NADPH gen'd in chloroplast cancross

chloroplast membrane

Photosystems

LHCs - l ight harvesting complexes

exciton quantum of energy passed from excited molecu

to another

spatially distinct, located in thylakoid membrane (lamella

o PSlllocated on appressedside

o PSI located on nonappressed side

chlorophyll Mg2+ held in porphyrin ring

o P68 and P7 specialized chlorophyll molecules located

reaction centers2 photosystems required to evolve O2 (oxygenic

photosynthesis)

o plants, algae, & cyanobacteria - contain both PSI and PS

o purple bacteria and green sulfur bacteria w/ only 1

photosystem, cannot evolve O2

Photosystem 11- Type II (Pheophytin-QuinoneJ Reaction Center

most similar to the bacterial reaction center

higher energy to excite (shorter wavelength)

Structure

OEC (Oxygen-evolving/water splitting complex)

o 2 H20 -> 4H+ + 4e- + O

2

o passes electrons to P680

o Manganese center/complex - binds water

cytochrome b6-f complex - pumps H+ into thylakoid

space

pheophytin - chlorophyll lacking central Mg2+ ion

P680 -> pheophytin -> Po,. ->PQB (plastoquinones) -> cytochrome

b6f complex ->PQsH2-> cytochrome f -> r a t a i f u c ~ n -> PSI~ ~ ~ ~ ~Photosystem 1- Type I Fe-S) Reaction Center

does not contribute to proton gradient

generates NADPH NADP+ from stroma)



-> A0 (Ao-) -> A1- (phyllquinone) -> 3x F e S ~ -> Fd (ferrodixin)+

+ H+

photophosphorylation

2 photosystems working in serial

-> plastoquinone -> cytochrome b6-f complex -> plastocyanin ->

-> ferredoxin -> ferredoxin NADP reductase

electrons cycle thru PSI reaction center

ATP formed w/ no net NADPH or O2

PSI -> cytochrome b6f complex '

accessory pigments that extend light absorption range

protect against damaging photochemical reactions reactive

oxygen species

8 carotene red-orange isoprenoid

lutein yellow blue light absorption

antennae of PSI of cyanobacteria red algae

proton pump only 247 amino acids long

sensory system

- allow plants to respond to light

phytochromes - cytoplasmic ser/thr kinases red ligh t

phototropin - blue light

cryptochromes - flavorproteins sensitive to blue light

CYCLE PHOTORESPIR TION

cyCle (carbon-fixation/assimulation)

- occursTn chforoplaststromaNADPH + 9 ATP + water -> glyceraldehyde 3-phosphate

2 -7-7-7 glyceraldehyde 3-phosphate)

Reactants Products

(3) CO2 + (3) ribulose 1,5 ~ (6) 3 ~ ~ o : ~ ~ ~ g l y c e r a t e1 bisphosphate

(6) 3-phosphoglycerate + ~ (6) 1,3-bisphosphoglycerate +

2 (6)ATP (6lATP

(6) 1,3- ~ Glyceraldehyde 3-phosphate +

3 bisphosphoglycerate (6) NADP+ + (6) i~ ? f>Jt\/ f

(5) glyceraldehyde 3- ~ (3) ribulose 5-phosphate + (2)4 phosphate i

(3) ribulose 5-phosphate + ~ (3) ribulose 1,5-bisphosphate

5 (3)ATP (first stable product)

(8 enzymes)

to plants)

to plants)

pentose phosphate pathway calvin cycle

Connects pentose phosphate pathway to glycolysis in anima

necessary for production of NADPH

fructose 6-phosphate + glyceraldehyde 3-phosphate ~ - 7erythrose 4-phosphate + xylulose 5-phosphate

sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate

ribose 5-phosphate + xylulose 5-phosphate

Light Regulation of Calvin cycle·

disulfide bondsformthat immobilize catalytic activity

Enzymes

Reductiono ribulose 5-phosphate kinase

o glyceraldehyde 3-phosphate dehydrogenase - prevents

hexose synth competing w/ glycolysis in dark

Regenetation

o fructose 1,6 bisphosphatase

o sedoheptulose 1,7 bisphosphatase

RuBisCO (ribulose 1,S-bisphosphate carboxylase/oxygenase)

substrates - 0 2 C02 ribulose l,S-bisphosphate

requires 2 CO2 for normal function (1 t

as substrate, 2nd

for

binding site of metal ion cofactor)

Photorespiration

use of O2 instead of CO 2 in Calvin cycle

caused when CO2 is in low concentrations

wasteful use of energy w/o useful products

involves chloroplasts, peroxisomes, and mitochondria

rubisco's oxygenase activity

o ribulose l,5-bisphosphate 7 3-phosphoglycerate +

phosphoglycolate

carbons salvaged in glycolate pathway releasing

previously fixed CO 2

C4 plants (spatial separation)

isolates Calvin cycle to maximize CO 2 concentration(minimizing energy water loss of photorespiration)

mesophyl l cells

o CO 2 + phosphoenolpyruvate (PEP) -> oxaloaceta

-7 malate - to bundle-sheath cells)

• pyruvate phosphate dikinase - pyruvate 7 PEP

• phosphoerolpyruvate carboxylase CO

phosphoenolpyruvate (PEP) 7

oxaloacetate

bundle sheath cells

o malate 7 CO 2 + pyruvate

• CO2 -7 Calvin cycle

•pyruvate + Pi + ATP -7 AMP + PP

i+ PEP

(to mesophyll cells)

CAM (Crassulacean acid metabolism) plants (temporal separation

-night - CO 2 fixed into malate stored in vacuoles

-day stomata closed, malate source of CO2

dihydroxyacetonephosphate

formed in stroma transports ATP out of chlo-roplasts (ATP

cannot be exported on its own)

transported by the Pi-triose phosphate anti por ter

converted by glycolytic enzymes to 3-phosphoglycerate,

generating ATP in cytosol



3-phosphoglycerate reenters the chloroplast, completing the

cycle

turned into starch in stroma

turned into sucrose production in cytosol

amylose al-4linked planar polymer of glucose

amylopectin - alA link highly branched polymer of glucose,

al-6 branch every 24-30 residues

synth'd in chloroplasts amyloplasts

o temporary storage chloroplasts of leaf cells

o long-term storage amyloplasts of colorless parts (seeds,

roots, tubers)

starch synthase - adds glucose to reducing end (donated by

ADP-glucose)

o Glucose I-phosphate + ATP j ADP-glucose

o ADP-glucose -(starch synthasej-j starch

ADP-glucose pyrophosphorylase

o regulates starch synthesis

o activated by 3-phospholycerate / inhibited by Pi

synth'd in cytosol of leaf cells

2 steps from U P ~ g l u c o s e + fructose I-phosphate

UDP-glucose -(cellulose synthase)-> cellulose

synthesis takes place in terminal complexes (rosettes)

rosettes simultaneously produce 36paraileiceHulosechains

DRI

catabolic processes (fatty acid oxid,) occur in the mito. matrix

inner membrane invaginations

- transfer across outer mem

- transfer acrOss inner mem - aided by memb potential

- helps jl-barrels fold in outer membrane

complex - moderates insertion of inner mem proteins that are

ndrial Hsp70 - acts as motor to pull precursor protein i nto

f?,;F I,,,

nsfer sequence prevents further translocation across inner

genome

Human mitochondria encode own ribosomal RNAs

-Plant mitochondria encode 5S mitochondrial rRNA, uses

standard genet ic code

membrane

most lipids imported modified rather than synthesized

high percent of lipids converted into cardolipin 4 fatty acid

tails)

impermeable to NADH (shut tle systems carry in reducing

equivalents)

proton gradient active transports:

o in pyruvate, Pi, ADP

o out ATP out

3 protein types

o electron transport chain

o ATP synthase

o metabolite transport proteins

Electron Transport (Respiratory) Chain

# Name Reaction e' carriers Notes

+

ADH dehydrogenaseNADH -7

FMN, FeSubiquinone

succinate-Q reductase / succinate -7FAD, FeS

not involved

dehydrogenase ubiquinone proton gradiubiquinone - mobile carrier (synth'd from benzoquinone [tyrosine

isoprene {acetyl-CoAl

Cytochrome b l

hemes,ill , complex ubiquinone -7 Rieske - 2 H

CoQ cytochrome c cytochrome cFeS, Rieske

instead of C

oxi do red ucta seISP

cytochrome c - mobile carrier - heme group, role in apoptosis

IV cytochrome coxidasecytochrome c -7 hemes;

O2 CUA CUB

ATP synthesis

Mitchel l chemiosmotic hypothesis - e' transport generates

proton-motive force that provides energy for ATP synthesis

Location - mito. Matrix, chloroplast stroma, cytosol in prok

ATP synthasome

o ATP synthase

• Fl - ATPase head + rotor stalk

• Fo - transmembrane H+ carrier / proton pore

o adenine nucleotide trans/ocase ATP/ADP antipor ter)

exchanges ATP/ADP ADP availability limits synthesis

o phosphate translocase symports H2P04- H+ into

matrix

o therrnogen (uncoupling protein) - energy of oxidatio

not conserved as ATP, but dissipated as heat

Shuttles

malate-aspartate shuttle (liver, kidney, heart) 2.5 ATP

NADH + oxaloacetate j NAD+ + malate j oxaloacetate +

NADH

reducing equivalents from cytosolic NADH transferred to

malate (malate transported replenishes NADH)

ions from malate, not malate transported across inner mito

memb:-r

g y c e r o I 3 ~ p h o s p h a t e shuttle (skeletal muscle + brain) 1,5 ATP

NADH to Complex III

converts reducing equivalents directly to ubiquinone in inn

mito. memb.

Oxygen Radicals

superoxide dismutase - antioxidant in cytosol, mitochondria

extracellular 2 02 + 2H+ 7 H20 2 +O2)

glutathione peroxidase reduce lipid hydroperoxides hydrogen

peroxide H 20 2 j H20)

CYTOSKELETON

ctin

Form



examples: contractile ring (myosin II), muscle (myosin II),

stereocilia, microvilli

bindsATP/ADP

o Tform grow (ATP)

o D form - shrink (ADP) [cofilin - causes D-form actin

disassembly]

plus end - faster polymerization & depolymerization

actin capping - dramatically lowers critical concentration &

increases growth rate

Nucleated at cell cortext (just beneath PM)

nucleates most effectively when bound to existing actin

filament

branch from preexisting filament by ~ 7 0 °nucleate actin growth by capturing two actin monomers,

subfamily of Ras, regulate many aspects of actin & MT

ARP & formin)

bound to bound to GDls (guanine nucleotide dissociation

factors) when inactive

play part in cell proliferation, division, gene expression, &

apoptosis

Cdc42 - affects filopodia

Rac1 affects lamellipodia

RhoA - affects stress fibers

ERM family - mediates interactions between actin & PM

dystrophin - connects muscle fiber to EM thru PM

spectrin dimers

Actin arrays

contractile bundle - t1-actinincrosslinks

gel-like network - filamin crosslinks - important for cell

motility

tight parallel bundle - fimbrin crosslinks

proteins

myosin superfamily

o Structure:

• 2 heavy chains a-helixes coiled-coi l + 4 light chains

(51 fragment - myosin head)

• N-term - head/motor domain

• C-term tail

motor activity Regulated thru pp'tion

embrane protrusions

Activity

Types

actin filaments bend

arp2/3 nucleation of new filaments

actin depolymerization & polymerization

Filopedia - 1D projections

Lamellipodia - 2D - sheet-like extensions

Pseudopodia - 3D

Regulation

Rac superfamily (related to Ras) stims formation of

lamellipodia & stress fibers

o Activated by growth factors

Form

Consist of protofilaments made up of tubulin

a-tubulin & - ~ - t u b u l i n dimerize

o a oriented toward - ) , ~ oriented toward (+)

o u-boundGTP intra linksdimer (cannot be lost)

o ,-bound GTP inter-linksdimers (can be lost)

Microtubule Nucleation

critical concentration of tubulin, energy (37"C), & GTP

MTOC (microtubule organizing center)

y-TuRC y ~ t u b u l i n ring complex) nucleates microtubule

centrosome (single well defined MTOC in animals)centrioles - cylinders arranged in L shape at centrosome

center

centrosome matrix/pericentriolar material

spindle pole body (MTOC in fungi & diatoms)

imbedded in nuclear membrane

microtubules nucleate ll aroundnuc/eus in plants

Microtubule motor proteins

Kinesin mostly toward +) 2 heavy chains, 2 light chains

one family C-term head, walks toward (-)

o Hand over hand motion - never completely dissociate

o Nonclaret disjunctional(NCD) toward (-) kinesin

Dyneins - toward (-) - 2-3 heavy chains + variable

intermediate & light chains

o Largest & fastest molecular motors

dynactin protein complex

links cargo (organelles, chromosomes) to dynein & kinesin

Associated proteins

M Ps (microtubule-associated proteins) bind alongside MTs

mediate interactions b/w MTs & other components

Tau & MAP2

plectin crosslinks actin, MTs & intermediate filaments

catastrophe factor increases chance of microtubule catastrophe

Mitotic Spindle

Centrosome - spindle pole

MTs

o astral interact w/ cell cortex - dynamic instability

o kinetochore - attach each chrom to each spindle pole

o interpolar - hold halves of spindle together by overlapping

Kinetochores attach MTs ~ 1 5 ) to chromosomes

in animals, position of contractile ring determined by signals from

anaphase spindle

poleward flux - net addition of tublin to +), loss at Haverage lifetime of microtubules during mitosis is 60 90 seconds

Ran-GTP & GEF bound to chromatin, stabilize MT spindles

Cilia flagella

Axoneme 9 doublet MTs (A-full, B-partial) arranged arou

2 MTs

Dynein responsible for MT sliding during flagellar bendin

Flagella = bacterial flagella (made up of flagellin, not MTs)

Basal bodies kinetosome) firm root of most cilia & flagel

Neurons - single MTs do no extend over the length of

axons/dendeites overlap

Dendrites - MTs of mixed polarity

Intermediate filaments



_ tetrameric,disassembly triggered by subunit phosphorylation

keratins - epithelial cells (hair, horns, etc)

o high % of glycine, alanine, & cysteine (14%)

o heterodimers of type I (acidic) & type II (neutral/basic)

o important in cell adhesion (in desmosomes)

desmin - heart muscle

neurofilaments - highly concentrated in axons

Y LE

yokinesis - nuclear div w/o cytoplasmic

of H1 & Rb limit replication to once per cell cycle

point - replication commitment (during GI )

checkpoint - triggers events leading to chromosome

sister-chromatid separation (unattached kinetochores inhibit

separation)

- specialized nondividing state

terminally differentiated Go - cell-cycle control system

completely dismantled

in G1

assembly of pre-replication complexes at origins

degradation of S-phase cyclin-Cdk inhibitor

activation of pre-replication complexes

APC/C activity

o degradation of securin

o degradation of mitotic cyclins

G1Cdc1 binds to APC/C - prevo inhibited by pp by M-cyclin-Cdk

Cdc1-APC/C activator

CKI accumulation + decr. cyclin expression suspend Cdk activity

S-cyclins - trigger progression through star t/rest riction point

- hours in mammals

synth increases during G2 & M due to increased transcription

Activated by Cdc25 removing inhibitory pps (by Wee1)

- activated by dephosphorylation at onset of mitosis

activates Cdc25 & inhibits Wee1

Induces assembly of mitotic spindle

Insures at tachment of sister chromatids to both poles of

spindle

Triggers chromosome condensation

Promotes breakdown of nuclear envelope (in animals)

Aided by pole-like kinases & aurora kinases

APC/C)

ubiquitin ligase

Activating subunits

Cdcl activation during late mitosis & early G1

o

o

inactivates Cdks & destroys geminin (allowing fo

pre-RC assembly)

pp d by M-Cdk allowing Cdc20 to bind

Cdc20 - activating subunit of cyclosome dur ing anaphase

(increased transcription as cell a'pproaches mitosis)

o Targets securin, S & M-cyclins for destruction (lo

of most Cdk activity)

dk Regulation

phosphyrlation of Cdk-subunit

o Cdk-act ivating kinases CAK) - fully activates cyc

Cdk complexo Cdc25 - removes i nhibitory pp'tion, increaSing C

activity

Binding of CKls (Cdk-inhibitors)

o Wee1 inhibits cyclin-Cdk complex

Proteolysis of cyclins

Changes in gene expression of Cdk regulators

Securin - binds to and inhibits separase - protease which cleaves

cohesion

Control of Cell division & Growth

Mitogens - stim Cl/S-Cdk activity & cell divisiono Ras GTPase) - activates MAP kinase cascade - Myc &

other gene reg. proteins

o G1 > G1-Cdk -> E2F proteins - G1/S-cyclins / S-cyclins

DNA synthesis

Growth factors - stim cell growth (increase in mass) -

promotion of synthesis & inhibition of degradation

o TOR - stims S6 kinase, elF4E (initiator factor)

o Promotes gene expression of ribosomal subunits

Survival factors - suppress apoptosis

DNA damage-7 ATM/ATR kinase -7 Chk1/Chk2 kinase -7 p53 -7 p2

binds G1/S-Cdk & S-Cdk

Mdm2 - degrades unphosphorylated p53

Stages o M Phase

(1) prophase - chroms condense, spindle assembles bin

centrosomes

(2) prometaphase - breakdown of nuclear envelope, chroms atta

to spindle

(3) metaphase - chromosomes align at equator, sister chromatid

attached to opposite poles

(4) anaphase - chromosome segregation - synchronized separat

of sister chromatids

anaphase A - shortening of kinetochore MTs & movement o

chromosomes along MTs towards polesanaphase B - sliding & elongation between interpolar MTs,

pulling force of astral MTs pull centrosomes apart

(5) telophase - chroms arrive at poles & decondense, nuclear

envelopes develop

o depp of proteins responsible for reassembly of nucleus

(6) cytokinesis - contractile ring (actin + myosin) divides cytoplas

Prophase I

leptotene - homologs condense & pair, recombination begins

zygotene - synaptonemal complex develops at recombination site

pachytene - assembly complete, homologs synapsed along enti re

length (can persist for days or longer)



disassembly of synaptonemal complexes & chromosome

- transition to metaphase I, thromsdetach from nucleus

- period lw meiotic divisions

b nd - marks site of cell wall assembly in plants

- precursor to cell wall formed on interpolar MTs in

OPTOSIS

- inactive precursors proteolytica lly cleaved by other

cleavesd in two spots large & small subunit - dimerize & form

tetramers

Prodomains I caspase recruitment domain - discarded

~ y s t e i n e active site & cleave at aspartate c-asp-ases)

Init iator procaspases - initiat ion executioner procaspases

Executioner procaspases cleave other EPs & target proteins,

cleaves protein that inhibits endonuclease

lAPs inhibitors of apoptosis) - inhibit caspases

Anti-lAP - bind to lAP preventing their interference wi

caspases

p thw y

extracellular signaling proteins bind to death receptors

Fas receptor binds Fas ligand

procaspases bind to death domains of Fas forming DISC

(death inducing signal complex)

FLIP - competes w/procaspase for binding on death domains,

lacks proteolytic activity

release of mitochondr ial proteins into cytosol some activateproto ytic caspase cascade

Cytochrome c - binds to adaptor protein Apafl (apoptotic

protease activating factor-i

Apoptosome heptamer of cytochrome c +Apafl

proteins regulate release of cytochrome c

BHl-4 anti-apoptotic, binds 1-4 (Bcl2 homology 1-4)

Bcl2 & Bcl-XL on cuytosolic surface of mit.mem.

BH123 pro-apoptotic, binds 1-3 - aggregate to pump out

cytochrome c

Bak - bound to cytosolic surface of mito.mem.

Bax - cytosol, transferred to Mito.mem. when active

BH3-only pro-apoptotic, inhibit anti-apoptotic

proteins

P53 - stims expression of BH3-only genes

stim expression of Bcl2

Uninhibit Bcl2 from BH3-only Bad, etc)

Uninhibit lAPs (pp'ing anti-lAP (Hid, etc)

- cleaves sphingomyelin into ceramide,

to be part of apoptosis

- condensed appearance of DNA indicating apoptosis

Cell swells wi water & ruptures

turmor necrosis factor 7 diacylglycerol 7 sphingomyelin

breakdown

CELL DHESION

Cell Cell Anchoring Junctions

Junction name Internal External

adherens cadherin actin

desmosomes cadherin intermediate filament

Cadherins - in animals, not plants or bacteria

- Depender:lt on ci+- Generally like-like binding

adhesion belt - cad herins anchor proteins + actin

Desmosomes contain the transmembrane cadherin proteins

desmoglein and desmocollin

Occluding Junctions

I Junction n me Organisms Proteins

I Tight junctiorl vertebrates claud in & occludin

I Septate junct ion invertebrates

fectins proteins that can bind carbohydrates, read sugar code

- sefectins - mediate cell-cell recognition

Immunoglobulin superfamily proteins mediate ci+ independent

cell-cell adhesion

- ICAMs intracellular cell-adhesion molecules

VCAMs - vascular

NCAMs neural

Junctiional complex - series of unctions between epithelial cells

tight, adherens, desmosomal

Signal-relaying junctions

Gap junctions - connect cells electrically & metabolically

o Connexins

o Coordinate activities of adjacent cells through sharing of

metabolites & ions

• Synchronize muscle contractions

• Important part of embryogenesis

o Permeability regulated - dopamine & Ca2+ (reduce or clos

Plasmodesmata - desmotubule - continuous wi ER

Chemical & Immunological synapses

o Scaffold proteins - important for cell-cell adhesion at

synapses

Cell polarity - dependent on membrane-associated proteins: Par3

Par 6 (scaffold proteins) & atypical protein kinase C aPKC)

Cell Matrix Anchoring Junctions

Junction name Internal External

actin-linked matrix integrin actin

adhesions

Hemidesmosomes integrin intermediate fila m

Integrins

- mUltiple forms from multiple genes

- a + 3 subunit clamps onto matrix

talin - binds integrin to actin



ronectin - glycoprotein that aids in matrix.-cell interactions

through Integrins

dependence - cell proliferation & survival tied to binding

adhesion kinase FAK) - cross-pp - docking site for

tyrosine kinases

Number of focal adhesions regulate level of adhesion/motility

Matrix

Synth & maintains EM

can differentiate into

o bone cell (osteoblast/osteocyte)

o fat cell (adipocyte)

o cartilage cell (chondrocyte)

o smooth muscle cell

EM

rich in nitrogen-containing polymers

excreted largely by fibroblasts

o molecular filter - proteoglycan key

o cell segregation

o scaffold

o regeneration - tough, often remains inact, guides repair &

regeneration

o fibrous proteins (usually glycoproteins)

o laminin - 3 chains, shaped like cross

o Type IV collagen - ropelikesuperhelix

o nidogen

glycosaminoglycans GAGs) + core proteins

EM

(Glycosaminoglycan) - unbranched polysaccharides composed

repeating disaccharide units 1 an amino sugar)

o Hyaluronan - simplest GAG, spun out directly from cell

surface by embedded enzyme complex

major proteins of EM

exported from ER as folded triple helix

repeating tripeptides (glycine-x-y) rich in glycine, proline &

hydroxyproline

mature collagen does not contain cysteine or disulfide bonds

o fibrilla r collagens - Type I principle coHagen of skin & bone

o Fibril-associated collagens -IX & XII- retain propeptides

organize fibrils

o Network -forming collagen - IV

o Anchoring fibrils - VII

tic fibers elastin - hydrophobic

Covered by sheath of microfibrils that act as scaffolding

fibrillin

lfate - present in all animal cells

common sugars:

o N-acetyl-O-glucosamine

o O-glucuronic acids (O-xylulose 5-phosphate 7 pentose

phosphate pathway

o L-iduronic acid

Matrix degradation

Matrix metalloproteases - depend on bound Ca2+or Zn

2+

Serine proteases - highly reactive serine active site

Plant Cell walls

almost no nitrogen

microtubular - cortical array determines orientation o

cellulose synthase

Primary cell walls

thin, extensible walls of newborn plant cells

o cellulose microfibrils interwoven w/ network of pectic

polysaccharides

o Pectins - negatively charged branched polysaccharide

Secondary cell walls

o rigid wall formed by deposit ing new layers of matrix inside

old ones

o Lignin network of phenolic compounds

Calmodulin (CaM) - calcium-binding protein - binds 4 Ca2+

responsible for regulatory ability of ci+F h nd - helix-turn-helix found in Ca-binding proteins

major classes

fibronectin Ig-superfamily CAMs, P-selectins

CaM kinases I-IV Ca2+/calmodulin dependent protein kinases)

serine/threonine kinase

phosphorylates self (memory)

DEVELOPMENT DIFFERENTIATION

Hoxcomplex

important switches that control cell identities by controlling

transcription target genes

carries permanent record of positional information

controls anteroposterior axis

Mammals have 4 Hox complexes

Morphogens

long-range inducers that exert graded effects

Hormonal signals that shape development, imposing a

pattern a whole field of cells

Induces graded responses, new gene expression, differentiat ionevents, & change in cell fate

Embryological Deve lopment

Concepts

Synctium - cell w/ multiple nuclei

Cel/u/arization subdivision of synctium so that each cell has only

one nucleus

Structures

embryonic disk ectoderm + endoderm

Blastula - hollow ball of cells, with an internal fluid-filled cavity

inner cell mass gives rise to extra-embryonic structures

hypoblast & trophoblast



- invagination that circle vegetal pole

Organizer (Spemann'sOrganizer) dorsal lip of blastopore

which organizes formation of main body axis

o n o t o h o r d ~ o u t g r o w t h of organizer-that defines central

body axis

ulation - transformation of simple ball or hollow sphere of cells

structure with a gut

3 germ layers formed

ectoderm - exterior, precursor of epidermis and nervous

system

o neural tube

endoderm interior, precursor of gut and its appendages' (lungliver)

mesoderm cells blw ectoderm endoderm; precursor of

muscles, connective tissues, etc.

o notochord defines primitive axis of embryo

o somites segments (cohesive groups of celis, separated

by clefts)

• form along neural tube dermis, skeleton, muscle

(heart)

serially repeating segments

groups of cells that are set aside, apparentlytiated, in each segment of the larva

n clock repetitive alternating pattern of gene

strategy for generating a progressively more

of few basic themes

genes

gap - divides embryo into broad regions

pair-rule segment polarity defines segment stripes

homeot ic - specifies which organs appendages develop

polarity - established as early as oogenesis in some species

Left-right polarity

o controlled by odal in embryo

Anterior-posterior patterning

o in limb bud Shh gradient

o in developing spinal chord - BMP, netrin hh

id - anterior

os - posterior

Carcinomas - epithelial cells

Sarcomas - connective tissue

Leukemias - blood or bone marrow

Lymphomas lymphocytes

of cancer growth

more self-sufficient

o high glucose uptake metabolism

o insensitive to anti-proliferative signals

o less prone to apoptosis

o induce help from stromal cells

o induce angiogenesis - format ion of new blood vessels

o produce telomerase or other method to stabilize

chromosome length

o reduced growth factor requirement

altered morphology

o metastasize

o genetically unstable

o loss of actin microfilaments

o defective differentiation

o defective cell cycle braking

protein kinases are found to be overactive in cancer cells

Types of carcinogenstumor initiators - damage DNA

tumor promoters - do not damage DNA

Cancer-critcal genes

proto-oncogenes - cancer risk from over activity - fos, myc

ras often g rowth factors and growth factor receptors

tumor suppressor genes - cancer risk from too little activit

DNA maintenance genes - mutat ion results in genetic

instability

Src - proto-oncogenic tyrosine kinases

Ras - g protein; mutated in 1 in 5 cancersRb - tumor suppressor

Myc - transcription factor I activator

Cancer Diagnosis

Intermediate filaments - may be used to diagnose tissue of origin

mes test assay to assess mutagenic potential

IMMUNOLO Y

pathogens

obligate pathogens can only replicate inside the body of their ho

and are calledfacultative pathogens replicate in wate r or soil, cause disease if

they encounter susceptible host

opportunistic pathogens normally harmless, cause disease in

injured or immunocompromised host

vaccination enhanced function of B cells cytotoxic T cells

autoimmune disease subverts immune system by destroying

helper T cells

transformation uptake of naked DNA

transfection introduction of foreign DNA by non-viral method

transduction foreign DNA introduced via phage vector

conjugation genetic material transferred unidirectionally from

bacterial donor to recipient

recipient F

donors

o F piliated w conjugative plasmid

o F - conjugative plasmid contains chromosomal genes

o Hfr - conjugative plasmid incorporated into chromosome

rolling circle (sigma) replication

Bacteria

classified by shape (rods, spheres, spirals) Gram-staining

properties





o activate endothel ial cells - cause monocytes & neutrophils

in the blood to adhere

o chemokines - positively charged cytokine that directs

migration of monocytes & neutrophils to infection

. TH2 cells

secrete cytokines interleukins 4 and 10 lL4 and ILlO)

o stim B cells to make antibodies (lgM, IgA, IgE, & some IgG)

cytotoxic T Cells

induces apoptosis in infected cells

o Fas ligand - binds receptor initiating caspase

cascade

per/orin - pore-forming protein r e l ~ a s e d by cytotoxic T censcross-presentation - activation by uninfected dendritic cells

regulatory T cells - suppress activity of other T celis

large role in humoral immune system

make receptor & secretory antibodies (expanded ERs)

plasma cells

memory B cells - form following primary infection

megakaryocytes

o responsible for platelets

o extraordinari ly large w highly polyploid nucleus,

remains in bone marrow

Receptors

TCRs) - antibody-like heterodimers

immunological synapse - interface lw antigen-presenting

cell & lymphocyte

diversity thru D-to-D joining, junctional imprecision, & N-

nucleotide addition

receptor TLR) - pattern recognition receptors that triggers

immune response to pathogens (microbial DNA, etc.)

proteins - exclusively intracellular pattern recognition

major histocompatibility complex)class I MHC - on all cells, display peptides from cytosolic

proteins

o a chain & ~ 2 m i c r o g l o b u l i n CD8)

o usually bind to cytotoxic Tcelis

class II MHC - macrophages, dendrit ic & B celis, display

endocytosed peptides

o usually bind to helper & regulatory T cells CD4)

Ig) - 2 heavy & 2 K & A light chains,

tail & Fab binding site

Primary classes

IgM - Il heavy chain - monomer or pentamer; first & major

antibody in primary immune response, activates complement

IgD - < heavy chain - monomer; second class to appear on

surface of B celis

Secondary classes

IgA - a heavy chain - monomer, dimer or trimer; principle class

in secretions (saliva, tears, milk)

IgE - E heavy chain - monomer; allergic response, binds mast

cells in tissues & basophils in blood (allergic reactions)

IgG - Y heavy chain - monomer; major antibody in secondary

immune response, most abundant in blood; activates

complement

o tail binds to Fc receptors on tnacrophages & neutrophils

class switching - after stimulation, B cells switch from IgM & IgD to

other antibodies (does not involve l ight chains)

affinity maturation - antibody affinity for antigen increases over ti

polyclonal antibodies - produced by many different B cells

responding to one antigen that bind to different epitopes

monoclonal antibodies - identical antibody produced by identical

cell in response to one antigen

V D)J recombination

light-chain V region V + J 7 VJ)

-long V gene segment

- short J gene segment joining)

heavy chain V region 0 + J 7 OJ + V 7 VDJ)

0 gene segment diversity)

. - short J gene segment joining)

-long V gene segment

RSS recombina tion signal sequences) - before Js & after Vs

RAG recombination activating genes)

-cuts in gene segments & recombination signal sequences

- recruits rejoining enzymes (DNA double-strand repair)

junctional diversification - random loss and gain of nucleotides at

joining sites of antibody gene segments

allelic exclusion - choice between maternal & paternal chromosom

loci

activa tion-induced deaminase AID) - deaminates C to U in V regio

producing mismatches, repair produces mutations

somatic hypermutation - accelerated mutation rate in B celis

Self-tolerance

central tolerance

receptor editing self-reactive lymphocytes change their

antigen receptors

clonal deletion, self-reactive lymphocytes apoptose

peripheral tolerance

clonal inactivation (clonal anergy), self-reactive lymphocytesfunctionally inactivate

clonal suppression regulatory T cells suppress sel f-reactive

lymphocytes

METHO S

DNA librar ies

genomic DNA l ibrary- collection of plasmids containing genome o

organism (not necessarily in whole genes)

cDNA l ibrary- collection of plasmids containing cDNA only (DNA

complementary to mRNA), not full genome

o contains info on level of varying expression - some cDN

present in duplic it

o contains unin terrupted coding sequences

Mutation methods

Site-directed mutagenesis - DNA is cut, DI\IA is inserted

Oligonucleotide-directed mutagenesis - complementary segment

made w single base-pair mismatch

hybridization & replication result in site mutation

induced somatic mutations - modification of DNA in subset of celi

at late stage of development

genetic mosaic- organism subjected to induced somatic

recombination



fingerprinting - does not normally use DNA microarrays

number of tandem copies of a simple sequence repeat

uses PCR, restriction endonuclease digestion & southern

blotting

RFLP - restriction fragment length polymorphism

microdissection

use of laser to move organelles, chromosomes

- images from a series of focal planes are combined to

an in focus plane

(up to .2 11m)

confocal- excludes out of focus light

o multiphoton confocal- uses multiple photon absorption

for greater penetration

fluorescence microscope - uses fluorescent dyes

,. TlRF total internal reflection fluorescence) - use of indirect

light to image cell- visualization of single molecules

Transmission (TEM) (up to Inm (10 angstroms) for biological

specimens) [0.1 nm for non-biological]

o difficult preparation/fixing - fixed, dehydrated, resin-

embedded

o supercooled into vitreous ice (noncrystalline glass )

Scanning SEM) (up to 10nm res)

o 3D structure of surface (10nm res) - used for cells & tissues

not subcellular components

Techniques

immunogold - gold particles attached to antibodies

metal shadowing - heavy metal evaporated onto specimen

from oblique angle

o negative staining - additional step to invert image

crystallography - EM + electron diffraction analysis

cryoelectron - specimen can be viewed w/o fixation, staining,

drying (cryogenic container at -160C)

o cryostat - cuts thin slices of specimen

single-particle reconstruction - thousands of images of

identical molecules are arranged together

tomography- 3D through rotation of specimen in TEM and

combination of images

atomic force - allows manipulation of individual molecules w/

silcon probe

spectrometryprecise determination of the mass of a protein (mass used to

ID unknown protein)

determine length of hydrocarbon chains or position of double

bonds

Techniques

Matrix-assisted laser desorption ionization MALOI) - soft

ionization process used on biomolecules

Time of Flight TOFMS) - mass-charge ratio of ions used to ID

molecules

TOF/TOF - tandem mass spectrometer - change in mass of IDs

amino acid lost (leucine/isoleucine same mass)

Type I - cleave DNA at random sites (up to 1000 bp from

recognition site)

Type cleave DNA within known recognition sequence

Type cleave ~ 5 b p from recognition site (requires ATP)

isoschizomers - restriction enzyumes from different species w

same recognition sequence

peR (polymerase chain reaction)

single-stranded DNA primers

deoxynucleotide triphosphates (dNTPs)

dideoxynucleotide triphosphates (ddNTPs)

stringency-level of homology; fidelity & specificity of hybridannealing

fidelity - depends on temp, pH, [NaCI], & probe sequence

U:;R ligase chain reaction) - good for detection of alleles, point

mutations

Vectors

plasmids 5,000-400,000 bp - dif ficult to clone segments long

than 15,000 bp, mUltiple per cell

o cosmid - have euk & prok initiation sequences

phages 40,000-53,000 bp - ID by plaque formation

BACs (Bacterial Artificial Chromosomes) - plasm ds for clonin

DNA of 100,000-300,000 bpo Accommodates largest inserts (up to 300k bp)

o from F plasmid, 300,000 - Imil bp, 1-2 copes per

YACs (Yeast Artificia l Chromosomes) -l inear, telomere stabl

longer than 150,000 bp

o contain features necessary for propagation as an

independent chromosome (centromere, telmomeres, o

o Plasm ids for yeast transformation - up to 2,000,000 bp

o Not recircularized, remain as linear molecules

Shuttle vectors - plasm ds that can be used to transform

different species

Expression vectors - cloning vector w/ transcription &

translation signals needed for regulated expression

o plasmids designed to produce large amounts of mRNAo contains promoter , ribosome-binding site, & transcriptio

termination sequence

Viral vectors - used in animals for transformation

Delivery of non-permeable substances - vectors, etc)

micropipette injection - injection of DNA directly into cell

and/or nucleus

electropora tion - electric shock leaves bacterial cell membra

temporarily permeable to DNA uptake

membrane-enclosed vesicles

gold particles - shot into cells (DNA coated)

Fluorescence

GFP (Green Fluorescence Protein)

FRET (fluorescence resonance energy transfer)

cellular location of reactions

FACS (Fluorescent-activated cell sorting) - used to separate cells b

DNA content, size, surface markers

FRAP (fluorescence recovery after photobleaching) -laser beam

extinguishes fluorescence --recovery recorded

Electrophoresis

SDS (sodium dodecylsulfate) - determines purity and

molecular weights of proteins

o SDS treated proteins - smaller weight travels faster



o anomalous if protein contains carbohydrates in large %

isoelectric focusing - organic acids and bases establish pH

gradient across gel, target protein migrates to pH pi

two-dimensional use o fboth SDS& isoelectric focusing

pu Ised field gel electrophoresis - alternating voltage allows for

better resolution of large DNA molecules (>15-20kbs)

gel-mobility shift assay - detects DNA bound proteins through

retardation of movement

denaturing polyarylamide - used in DNA sequencing

electrophoresis; probing & hybridizaton in RNA/DNA,

y affinity in proteins \

allows detection of minor sample component and provides

estimated weight

target sequence usually twice as long as probe

o southern - DNA

o northern - RNA

o western (immunoblotting) - protein

reservoir - container

stationary phase column

mobile phase - solution to be run through column

effluent - solution passed thru columnTypes

cation-exchange - negatively charged stationary phase

hydrophobic hydrophobicity

gel fil tration / size-exclusion - separates according to size

(smaller proteins slowed down by pores/cavities)

affinity - functional groups in stationary phase bind to proteins

w/ varying affinity

HPLC (high performance liquid) - high pressure

chromatography that limits diffusional spreading maximizing

resolution

absorption or thin-layer - separates lipids of diff polarity

techniques

effective for detecting & quantifying hormones (can detect

very small amounts of specific material)

Techniques

RIA Radio Immuno Assay) quantifies amount of hormone in

sample

o incubate sample w/ hormone-specific antibody & purified

radiolabeled hormone

o unlabeled hormone competes & displaces radiolabeled

hormone from antibody

ELISA enzyme-linked immunosorbent assay) - screens for

presence of an antigen

Protein micro array / chip array of antibodies to ID proteinsCoimmunoprecipitation precipitation of complexed proteins

w/ targeted ones

epitope tag - use of known epitope to tag protein

fused cells w/ separate nuclei

- fused B lymphocyte w/ transformed tumor line cell) B

hocyte - molecular factory for monoclonal antibodies

/ dideoxy method - ddNTPs terminate DNA synth, creating

of various lengths sequenced thru electrophoresis

based on chemical modifications of DNA

Edman degradation label & removal of 1 AA at time (res: AA

sequenator automated Edman degradation

chromosome walking - sequencing thru cyclic creation of primers

Protein Sequencing

TOF/TOF - tandem mass spectrometer - change in mass of IDs

amino acid lost (leucine/ isoleucine same mass)

Sequence evolution (species relatedness)

Blosum62 (62% identical) - blocks substitution matrix, mathemati

analysis of genome evolution

Protein interactions

.Surface plasma resonance SPR) detects binding interactions by

ch,ange in resonance angle, good for timing of interactions

yeast two-hybrid system

IDs protein interactions w/ known bait ), co-activators & c

repressors

reporter genes easily assayed enzymes

Protein conformation

NMR spectroscopy - protein conformation

Genetic Analysisgenetic screen - genetic analysis of numerous individuals to ID

mutant allele responsible for mutant phenotype

pleiotropy - single gene influences multiple phenotypic traits

epistasis analysis - analysis of combinations of mutations in orde

determine order of gene action (epistatic - occurring prior)

complementation test - tests if 2 mutations are products of

mutations in one or two genes

Cre-Lox Recombination - gene knockout: lox sites - target gene, cr

recombinase remove it

protein synthesis - AAs - on polymer support

Gene Expression

DNA microarrays - rapid + simultaneous screening of thousands o

genes wIn species

probed w/ mRNA or cDNA ID what genes are being expres

Comparative genomic hybridization CGH) - DNA microarray

comparison to see what genes over/under expressed

DISORDERS TOXINS REAGENTS

DRUGS

Disorders

Huntington's disease - autosomal dominant disorder

Parkinson's disease - underproduct ion of dopamine

maple syrup urine disease lack of debranching AA deg. Enzyme

abnormal brain development & infancy death

anemia - hemoglobin deficiency

beriberi -lack of Vitamin B1 (thiamine) - involved in breakdown o

glucose

hypoxin -lowered oxygenation of peripheral tissues

Jaundice accumulation of bilirubin in blood b/c not enough

glucuronyl bilirubin transferase to process to bilirubin diglucuron

diabetes - failure of insulin secretion or action, fail to synth fatty

acids from carbohydrates or amino acids

acidosis & ketosis lowered blood pH caused by ketone bodies

(acetoacetate D-j3-hydroxybutyrate) in blood



sease - autosomal dominant disorder

kinson's disease - underproduction of dopamine

emia - hemoglobin deficiency

- lack of Vitamin B1 (thiamine) - involved in breakdown of glucose

-lowered oxygenation of peripheral tissues

ndice - accumulation of bilirubin in blood blc not enough glucuronyl bilirubin transferase to process to bilirubin diglucuronide

etes - failure of insulin secretion or action, fail to synth fatty acids from carbohydrates or amino acids

-lowered blood pH caused by ketone bodies (acetoacetate D - ~ - h y d r o x y b u t y r a t e in blood

(death caps) inhibits RNA polymerase

inhibit MT assembly by capping ends(blocks cell division), induces polyploidy in plants

CO, azide blocks electron transport from Complex IV (cytochrome oxidase) to O2

blocks elongation

DNP) dissipates proton gradient in mitochondr ia, eliminating ATP synthesis

inhibits CAC

inactivates EF-2, blocking elongation

(death caps) prevent actin disassembly

TAG) in skeletal muscle & liver can be toxic

purine analog that blocks purine breakdown, lowering uric acid levels

aspirin (NSAlDs) - inhib its cyclooxygenase COX), essential for arachidonate conversion to prostaglandins & thromboxanes

prednisone - inhibits arachidonic acid release by phospholipase A2 blocking prostaglandin formation

inhibit Na+,K+-ATPase (l [Na+], l [Ca2+]), increase contractile force

prevents elongation, inhibiting transcription

inhibits oxidation of CoQ by binding Complex III (cytochrome bCl complex)

binds 50S subunit, preventing translocation

blocks Fa (proton pore) of ATP synthase

causes premature release of protein chains, inhibiting synthesis

binds 30s subunit, preventing initiation

prevents synth of peptidoglycan cross-links in cell wall of gram-positive bacteria

prevents release of EF-G-GTP, blocking tranlocationinhibits peptidal transferase activity of bacterial ribosomes

binds 16S rRNA, blocking aminoacyl-tRNA

& performic acid oxidizes disulf ide bonds

chloride used in n-terminal AA analysis

used to cleave peptide chains at methionyl residues

used to modify cysteine side chains into thioethers, preventing disulfide bond reformation

& chloroform precipitate proteins out of lysate

Intercalating gents

o Planar molecules that lodge blw stacked DNA bases causing DNA pol to insert or skip bases

o proflavin acridine organe & ethidium bromide

base analogs

o less stable, causes mismatches

o 5-bromouracil & 2-aminopurine

alkylating agents cause mismatch by chemically modifying bases

o hydroxylamine GC 7 AT

spermatocyte -(mei-I)--7 sec. spermatocyte -(mei-II)--7 spermatid - spermatozoa (mature sperm)

-(rep.)--7 prim. oocyte -(meios is 1)--7 sec. oocyte -(mei-II)--7 oovid - ovum (mature egg)

PLP) - Schiff base + E-amino



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Documents

GRE BioChem