Gena Si Transcriptia La Pro Si Eucariote Lp2

7/27/2019 Gena Si Transcriptia La Pro Si Eucariote Lp2

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Lecture 3

Gene Structure, Transcription, &Translation

Reading: Chapter 4: 108-115; 118-131

Molecular Biology syllabus web site
http://a32.lehman.cuny.edu/webwurtzel/course/CURRIC99j.htmhttp://a32.lehman.cuny.edu/webwurtzel/course/CURRIC99j.htmhttp://a32.lehman.cuny.edu/webwurtzel/course/CURRIC99j.htm


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Typical Gene Structure

Promoter Coding Region

transcription

+1


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Prokaryotes

COORDINATED GENE

EXPRESSION: clustered

genes (operon)controlled by one

promoter and transcribed

as polycistronic mRNA

and encode multiple

gene products


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Eukaryotes

Interrupted genes(exons/introns)

Monocistronic mRNAs

Post-transcriptional

modifications (nuclear

encoded genes):

5 CAP

polyA tail

splicing


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Post-transcription addition of 5 CAP to nuclear encoded

eukaryotic mRNA


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3

5

5 3

Transcript Structure

5 untranslated 3 untranslated

AUGrbs

DNA

mRNA

ORF

Open Reading Frame

protein


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Requirements1. Enzyme: RNA Polymerase

2. DNA Template (3 to 5 strand)3. No primer required

4. Nucleoside triphosphates: ATP,GTP, CTP, UTP

5. Synthesis is 5 to 3

Transcription: RNA Synthesis


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Transcription: RNA Synthesis


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Translation: Protein Synthesis

Codons specify amino acids; positioning on

ribosome sets READING FRAME


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Copyright (c) by W. H. Freeman and Company

The roles of RNA in protein synthesis


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The three roles of RNA in protein synthesis

Three types of RNA molecules perform different butcomplementary roles in protein synthesis (translation)

Messenger RNA (mRNA) carries information copied from

DNA in the form of a series of three base words termedcodons

Transfer RNA (tRNA) deciphers the code and delivers thespecified amino acid

Ribosomal RNA (rRNA) associates with a set of proteins toform ribosomes, structures that function as protein-synthesizing machines


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The folded structure of tRNA specifies itsdecoding function

Figure 4-26


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Aminoacyl-tRNA synthetases activateamino acids by linking them to tRNAs

Each tRNA molecule is

recognized by a

specific aminoacyl-

tRNA synthetase


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Fidelity of protein synthesis

determined by:

Correct aminoacylation of tRNA

Codon-anticodon pairing


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Aminoacyl tRNA synthetases

-at least one for every amino acid

-for different codons have different synthetases-error correction lies in specificity of synthetase and tRNA. No

mechanism exists for error correction once tRNA is

mischarged and separated from synthetase

Double sieve mechanism for error correction

Synthetases have 2 sites: active site, hydrolytic site.

Amino acids larger than the correct amino acid are neveractivated because they are too large to fit into the active site.

Smaller amino acids (than the correct one) fit into the

hydrolytic site (which excludes the correct amino acid) and

are hydrolyzed.


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Nonstandard base pairing often occurs between

codons and anticodons


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Ribosomes: the macromolecular site for protein synthesis


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Translation

Initiation

Elongation

Termination


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Initiation

mRNA binds to ribosome

Selection of initiation codonBinding of charged initiator

tRNA (first amino acid)


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Initiation

Formation of 30S preinitiation complex30 S subunit (contains 16S rRNA),

mRNA, charged tRNA f-met, initiation

factors, GTP

+ 50S subunit (GTP hydrolysis)

Resulting in formation of the 70S initiation complex

fmet-tRNA is fixed into the P site

reading frame is now determined.


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Initiation


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Initiation of eukaryotic protein synthesis generally occurs at the5 end of mRNA but may occasionally occur at internal sites


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Initiation of prokaryotic protein synthesis generally

occurs at the Shine Delgarno site

The untranslated leader or 5 end of prokaryotic mRNAs contain

a ribosome binding site (rbs) orShine Delgarno site located

upstream of the AUG and complementary to the 3 end of the16S rRNA.

mRNA: 5 .AGGAGGU..AUG

3end of 16S rRNA 3 ...UCCUCCA..I I I I I I I


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Elongation

Peptide bond formation

Movement of mRNA/ribosome (translocation) so

each codon may be read


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Elongation

Occupation of A site by next tRNA

Peptide bond formed by peptidyl transferase enzyme

Uncharged tRNA-fmet in P site and dipeptidyl tRNA in A site

Translocation:

deacylated tRNA fmet leaves P site

peptidyl tRNA moves from A to P site

mRNA moves 3 bases to position next codon at A site

Requirements: Elongation factors and GTP hydrolysis


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Elongation


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Termination

Completedprotein is

dissociatedfrommachinery

Ribosomereleased

when termination codons are reached (UGA, UAA, UAG)

T i ti


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Terminationwhen termination codons are reached (UGA, UAA, UAG)

Peptidyl tRNA moves from A to P site

Release factors (RF) recognize specific stop codons

RF forms activated complex with GTP

Activated complex binds to termination codon and alters

specificity of peptidyl transferase

In presence of RF, peptidyl transferase catalyzes reaction of

bound peptidyl moiety with water instead of with freeaminoacyl tRNA

Release of polypeptide

Dissociation of 70S ribosome into 50S and 30S subunits.

S f P t i S th i


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Summary of Protein Synthesis1. Binding of mRNA to ribosome

2. Charged, amino-acylated initiator tRNA binds to P site ofribosome and is based paired through tRNA anticodon to codon

on mRNA

3. A second amino-acylated tRNA fillsA site and anticodon H-

bonds with second codon on mRNA4. Amino acids in P and A site are joined by a peptide bond.

tRNA in P site is released.

tRNA (with 2 amino acids joined) in A site moves to P site

A new amino-acylated tRNA moves intoA site by anticodon-

codon pairing

5. Step (4) is repeated until codon inA site is a stop codon;e tide is released.


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Post-translational Modifications

(Bacteria) removal of formyl groups (fmet)

removal of first few amino acids(aminopeptidase)

glycosylation (affects targeting, activity)

phosphorylation (by kinases)

S-S bond formation

Polypeptide cleavage

-removal of transit peptide upon organelle impor

-removal of signal sequence (ER secretion)-activation of enzymes

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Gena Si Transcriptia La Pro Si Eucariote Lp2