From Gene to Protein Chapter 17. What Is a Gene? The idea of the gene has evolved through the history of genetics We have considered a gene as – A discrete

From Gene to Protein

Chapter 17

What Is a Gene? • The idea of the gene has evolved through the

history of genetics• We have considered a gene as

– A discrete unit of inheritance – A region of specific nucleotide sequence in a

chromosome– A DNA sequence that codes for a specific

polypeptide chain

Proteins= link between genotype and phenotype

Flow of Genetic Information

• DNA= “directions” are encoded in nucleotide sequences– Sequences provide information for protein

synthesis• Gene expression= process by which DNA

directs protein synthesis– 2 stages: transcription and translation

Flow of Genetic Information• Central Dogma

– Concept that cells are governed by a cellular chain of command: DNA RNA protein

• RNA= link between genes and the proteins• Transcription= synthesis of RNA under the

direction of DNA– messenger RNA (mRNA)

• Translation= synthesis of a polypeptide, using information in the mRNA


DNA

RNA

Protein

DNA

mRNARibosome

Polypeptide

TRANSCRIPTION

TRANSLATION

TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome

DNA

mRNA

Pre-mRNARNA PROCESSING

(a) Bacterial cell (b) Eukaryotic cell

Nuclearenvelope

• Bacteria– Translation can begin before

transcription finishes mRNA molecule

• Eukaryotes– Transcription occurs in nucleus– Translation occurs in cytoplasm

Variation in Transcription and Translation

• Bacteria and eukaryotes differ in..– RNA polymerases– Termination of transcription– Ribosomes

• Archaea are prokaryotes, but share many features of gene expression with eukaryotes

Form = Function• High diversity of molecules due to arrangement

of monomers– DNA composed of 4 nucleotides – RNA composed of 4 nucleotides – 20 amino acids (monomers of proteins)

Form = Function• The flow of information from gene to protein is

based on a triplet code – Nonoverlapping, three-nucleotide codon

• Codons of a gene are…– Transcribed into complementary codons of mRNA– Translated into amino acids

Second mRNA base

Firs

t mRN

A ba

se (5

end

of c

odon

)

Third

mRN

A ba

se (3

end

of c

odon

)

UUU

UUC

UUA

CUU

CUC

CUA

CUG

Phe

Leu

Leu

Ile

UCU

UCC

UCA

UCG

Ser

CCU

CCC

CCA

CCG

UAU

UACTyr

Pro

Thr

UAA Stop

UAG Stop

UGA Stop

UGU

UGCCys

UGG Trp

GC

U

U

C

A

U

U

C

C

CA

U

A

A

A

G

G

His

Gln

Asn

Lys

Asp

CAU CGU

CAC

CAA

CAG

CGC

CGA

CGG

G

AUU

AUC

AUA

ACU

ACC

ACA

AAU

AAC

AAA

AGU

AGC

AGA

Arg

Ser

Arg

Gly

ACGAUG AAG AGG

GUU

GUC

GUA

GUG

GCU

GCC

GCA

GCG

GAU

GAC

GAA

GAG

Val Ala

GGU

GGC

GGA

GGGGlu

Gly

G

U

C

A

Met orstart

UUG

G

Cracking the Code• All 64 codons were deciphered by the mid-

1960s• Of the 64 triplets

– 61 codons represent amino acids– 3 codons are “stop” signals

• Genetic code– Redundant

• 1 amino acid coded by multiple codons

– Not Ambiguous• 1 codon = 1 amino acid

Cracking the Code

• Codons must be read in the correct reading frame– 5’ to 3’ direction

• 5’-AUGCUGGAACCGACCUGA-3’

Evolution of the Genetic CodeGenetic code is nearly universal…

From unicellular organisms….

..to multicellular animals

Evolution of the Genetic CodeGenes can be transcribed and translated after being transplanted from one species to another

Tobacco plant expressing firefly gene

Pig expressing jellyfish gene

Green sea slug expresses algal DNA• Green sea slugs (Elysia chlorotica) have functional

chloroplasts that carry out photosynthesis– Chloroplasts are taken up from food source= algae– Algae DNA incorporated into slug DNA

Transcription

• Occurs in Nucleus• RNA made from DNA directions

– Messenger RNA (mRNA)• Only one strand of DNA transcribed

– Template strand= 3’ to 5’– RNA made in 5’ to 3’ direction

DNA

RNA

Transcription• Enzyme= RNA polymerase• Purpose of RNA polymerase

– Separates DNA strands– Links RNA nucleotides together– Several dozen nucleotide pairs in length

• Section of DNA transcribed = transcription unit

DNA

RNA

Transcription

• mRNA= complementary to DNA template strand– same base-pairing rules as DNA,

with one exception• Uracil substitutes for thymine• Uracil-Adenine

• Eukaryotes– Primary transcript= Initial RNA molecule produced

by transcription• Additional processing needed to make final mRNA

DNA

RNA

Transcription: Initiation

• Promoter= nucleotide sequence signals binding site for RNA polymerase

• Transcription initiation complex= RNA polymerase II + transcription factors – Transcription factors= proteins control translation

of a gene by activating or preventing binding of RNA polymerase to promoter site

• A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes

Figure 17.8

Transcription initiationcomplex forms

3

DNAPromoter

Nontemplate strand

53

53

53

Transcriptionfactors

RNA polymerase IITranscription factors

53

53

53

RNA transcript

Transcription initiation complex

5 3

TATA box

T

T T T T T

A A A A A

A A

T

Several transcriptionfactors bind to DNA

2

A eukaryotic promoter1

Start point Template strand

Transcription: Elongation• RNA polymerase untwists the double helix

– 10-20 bases at a time– Eukaryotes Rate of Transcription=

40 nucleotides/second• Nucleotides= added to the 3 end of the

growing RNA molecule

Nontemplatestrand of DNA

RNA nucleotides

RNApolymerase

Templatestrand of DNA

3

35

5

5

3

Newly madeRNA

Direction of transcription

A

A A A

AA

A

T

TT

T

TTT G

GG

C

C C

CC

G

C CC A AA

U

U

U

end

Figure 17.9

Transcription: Termination • Prokaryotes

– Terminator= nucleotide sequence signaling end of gene, RNA polymerase separates from DNA

– mRNA does not need additional modification– Translation can begin immediately

• Eukaryotes– Polyadenylation signal sequence= RNA transcript

released 10–35 nucleotides past this sequence– Additional processing needed to convert transcript

into functional mRNA molecule– Processing occurs before leaves nucleus

Figure 17.7-4 Promoter

RNA polymeraseStart point

DNA

53

Transcription unit

35

Elongation

53

35

Nontemplate strand of DNA

Template strand of DNARNAtranscriptUnwound

DNA2

3535

3

RewoundDNA

RNAtranscript

5

Termination3

35

5Completed RNA transcript

Direction of transcription (“downstream”)

53

3

Initiation1

RNA Processing

• Enzymes in the eukaryotic nucleus modify RNA transcript before released to cytoplasm– Alteration of ends of the primary transcript – RNA splicing of interior portions of molecule

RNA Processing• Each end of a pre-mRNA molecule is modified

in a particular way– 5 end= modified nucleotide 5 cap– 3 end= poly-A tail

• Reasons for modifications– Facilitate export of mRNA from nucleus– Protect mRNA from enzymes in cytoplasm– Assist with ribosomal attachment to 5 end

RNA Processing• Most eukaryotic genes have introns between

coding regions– Introns= series of nucleotides in noncoding regions

of genes – Included in RNA transcripts

• Exons= coding regions of genes – Expressed when translated into amino acid

sequences• RNA splicing removes introns and joins exons

– Spliceosomes– Ribozymes

• End product= mRNA molecule with continuous coding sequence

What is the purpose of introns?

• Sequences may regulate gene expression• Genes can encode more than one kind of

polypeptide– Type of polypeptide depends on which segments are

removed during splicing– Alternative RNA splicing

• Advantage= Number of different proteins produced is much greater than its number of genes

RNA ProcessingSpliceosomes= proteins + small nuclear ribonucleoproteins (snRNPs)

– Recognize splice sites

RNA Processing• Ribozymes= catalytic RNA molecules

– Function as enzymes – Splice RNA– Discovery changed long-held belief that all biological

catalysts were proteins• Three properties of RNA enable it to function as an

enzyme– Form a 3-D structure

• Base-pair with itself

– Bases contain functional groups that act as a catalyst– Hydrogen-bond with other nucleic acid molecules

5 Exon Intron Exon5 CapPre-mRNA

Codonnumbers

130 31104

mRNA 5Cap

5

Intron Exon

3 UTR

Introns cut out andexons spliced together

3

105 146

Poly-A tail

Codingsegment

Poly-A tail

UTR1146

RNA Processing

Final Product


Transcription

DNA

RNA

Protein

Translation

Transcription Translation• Flow of information

from DNA to protein is the triplet code of bases

• mRNA moves out of nucleus and into cytoplasm

• Ribosomes attach to mRNA and translation begins

Translation•Ribosomes “read” code on mRNA to build polypeptide chain •Amino acids brought to ribosome by transfer RNA (tRNA)– Single RNA strand – ~80 nucleotides long

RNA

Protein

Ribosomes

TranslationMolecules of tRNA pair with a specific amino acid

• 3’ End= Amino acid attachment site

• Anticodon– Base-pairs with

complementary codon on mRNA

• Hydrogen bonds give tRNA its 3-D structure– L-shaped

Translation

• Accurate translation requires 2 steps:1. Match between tRNA and an amino acid

– Enzyme aminoacyl-tRNA synthetase

2. Match between tRNA anticodon and mRNA codon

• Wobble= flexible pairing at the third base of a codon – Allows some tRNAs to bind to more than one

codon

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P P P Adenosine

ATP

P

P

P

PPi

i

i

Adenosine

tRNA

AdenosineP

tRNA

AMP

Computer model

Aminoacid

Aminoacyl-tRNAsynthetase

Aminoacyl tRNA(“charged tRNA”)

Figure 17.16-4

Ribosomes• Facilitate specific coupling of tRNA anticodons

with mRNA codons– Two ribosomal subunits- large and small

• Proteins and ribosomal RNA (rRNA)

Ribosomes

• Two types of ribosomes – Free ribosomes= in the cytosol– Bound ribosomes= attached to the endoplasmic

reticulum (ER)– Both types are identical – Switch from free to bound

• Free ribosomes mostly synthesize proteins that function in the cytosol

• Bound ribosomes make proteins of the endomembrane system and proteins to be secreted from the cell

Ribosomes

• Polypeptide synthesis always begins in the cytosol

• Finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER

Ribosomes

• Three binding sites for tRNA– P site= holds the tRNA with the growing

polypeptide chain– A site= holds the tRNA with next amino acid to

be added– E site= exit site

Figure 17.17c

Amino end

mRNA

E

(c) Schematic model with mRNA and tRNA

5 Codons

3

tRNA

Growing polypeptide

Next aminoacid to beadded topolypeptidechain

Translation: InitiationBrings together translation initiation complex:

– mRNA– tRNA with first amino acid– two ribosomal subunits

1. Small ribosomal subunit binds with mRNA and initial tRNA

2. Small subunit moves along the mRNA to start codon (AUG)

3. Proteins called initiation factors bring in the large subunit

Translation: Initiation

• First tRNA attaches at P site• All other tRNA enter at A

site

Translation: Elongation• Amino acids added one by one to the growing

chain– Proteins called elongation factors

• Occurs in three steps– Codon recognition– Peptide bond formation– Translocation

• Translation proceeds along the mRNA in a 5 to ′3 direction′

Amino end ofpolypeptide

mRNA

5

E

Asite

3

E

GTP

GDP P i

P A

E

P A

GTP

GDP P i

P A

E

Ribosome ready fornext aminoacyl tRNA

Psite

Figure 17.19-4

Translation: Termination

• Stop codon in the mRNA reaches the A site of the ribosome

• A site accepts a protein called a release factor• Release factor causes the addition of a water

molecule instead of amino acid– Reaction releases polypeptide– Translation assembly separates

Translation• Polyribosome: multiple ribosomes translate a single

mRNA simultaneously– Enable cell to make many copies of a polypeptide very

quickly

Completedpolypeptide

Incomingribosomalsubunits

Start ofmRNA(5 end)

End ofmRNA(3 end)(a)

Polyribosome

Ribosomes

mRNA

(b)0.1 m

Growingpolypeptides

DNAtemplatestrand

TRANSCRIPTION

mRNA

TRANSLATION

Protein

Amino acid

Codon

Trp Phe Gly

5

5

Ser

U U U U U3

3

53

G

G

G G C C

T

C

A

A

AAAAA

T T T T

T

G

G G G

C C C G GDNAmolecule

Gene 1

Gene 2

Gene 3

C C

Modifications of Proteins• During and after synthesis, a polypeptide

chain spontaneously coils and folds into its three-dimensional shape

• After translation, many proteins must undergo modifications before becoming functional– Activated by enzymes that cleave them– Multiple polypeptide chains come together to

form a larger protein

Modifications of Proteins

• Targeted to specific sites in cell• Polypeptides destined for the ER or for secretion are

marked by a signal peptide

• A signal-recognition particle (SRP) binds to the signal peptide– Brings the signal peptide and its ribosome to the

ER– Signal peptide removed and protein enters ER for

transport

Effect of Mutations on Proteins

• Mutations = changes in the genetic material of a cell– Can occur during DNA replication, recombination, or

repair– Mutagens= physical or chemical agents that can

cause mutations• Point mutations= changes in one base pair of a

gene– Single change in a DNA template strand can lead to

the production of an abnormal protein

Wild-type hemoglobin

Wild-type hemoglobin DNA3

3

35

5 3

35

5553

mRNA

A AGC T T

A AGmRNA

Normal hemoglobin

Glu

Sickle-cell hemoglobin

Val

AA

AUG

GT

T

Sickle-cell hemoglobin

Mutant hemoglobin DNAC

Point Mutations

• Two general categories1. Nucleotide-pair substitutions2. One or more nucleotide-pair insertions or

deletions

Nucleotide-Pair Substitution

• Replaces one nucleotide pair with another pair of nucleotides

• Silent mutations= no effect on the amino acid because of redundancy in the genetic code

• Missense mutations= codes for incorrect amino acid

• Nonsense mutations= change an amino acid codon into a stop codon– Usually creates nonfunctional protein

Figure 17.24a

Wild type

DNA template strand

mRNA5

5

Protein

Amino endStopCarboxyl end

33

3

5

Met Lys Phe Gly

A instead of G

(a) Nucleotide-pair substitution: silent

StopMet Lys Phe Gly

U instead of C

A

A

A A

A A A A

A AT

T T T T T

T T TT

C C C C

C

C

G G G G

G

G

A

A A A AG GGU U U U U

53

35A

A A

A A A A

A AT

T T T T T

T T TT

C C C CG G G G

A

A

A G A A A AG GGU U U U U

T

U 35

Figure 17.24b

Wild type

DNA template strand

mRNA5

5

Protein


33

3

5

Met Lys Phe Gly

T instead of C

(a) Nucleotide-pair substitution: missense

StopMet Lys Phe Ser

A instead of G

A

A

A A

A A A A

A AT

T T T T T

T T TT

C C C C

C

C

G G G G

G

G

A


53

35A

A A

A A A A

A AT

T T T T T

T T TT

C C T CG

G

GA

A G A A A AA GGU U U U U 35

A C

C

G

Figure 17.24c

Wild type

DNA template strand

mRNA5

5

Protein


33

3

5

Met Lys Phe Gly

A instead of T

(a) Nucleotide-pair substitution: nonsense

Met

A

A

A A

A A A A

A AT

T T T T T

T T TT

C C C C

C

C

G G G G

G

G

A


53

35A

A

A A A A

A AT

T A T T T

T T TTC C C

G

G

GA

A G U A A AGGU U U U U 35

C

C

G

T instead of C

C

GT

U instead of AG

Stop

Insertions and Deletions• Additions or losses of nucleotide pairs in a gene

– Often has greater negative effect than substitutions• Frameshift mutation• Missing amino acid (loss of 3 nucleotides)

Figure 17.24d

Wild type

DNA template strand

mRNA5

5

Protein


33

3

5

Met Lys Phe Gly

A

A

A A

A A A A

A AT

T T T T T

T T TT

C C C C

C

C

G G G G

G

G

A


(b) Nucleotide-pair insertion or deletion: frameshift causingimmediate nonsense

Extra A

Extra U53

5

3

3

5

Met

1 nucleotide-pair insertion

Stop

A C A A GT T A TC T A C GT A T AT G T CT GG A T GA

A G U A U AU GAU G U U C

A TA

AG

Figure 17.24e

DNA template strand

mRNA5

5

Protein


33

3

5

Met Lys Phe Gly

A

A

A A

A A A A

A AT

T T T T T

T T TT

C C C C

C

C

G G G G

G

G

A


(b) Nucleotide-pair insertion or deletion: frameshift causingextensive missense

Wild type

missing

missing

A

U

A A AT T TC C A T TC C G

A AT T TG GA A ATCG G

A G A A GU U U C A AG G U 3

53

35

Met Lys Leu Ala

1 nucleotide-pair deletion

5

Figure 17.24f

DNA template strand

mRNA5

5

Protein


33

3

5

Met Lys Phe Gly

A

A

A A

A A A A

A AT

T T T T T

T T TT

C C C C

C

C

G G G G

G

G

A


(b) Nucleotide-pair insertion or deletion: no frameshift, but oneamino acid missing

Wild type

AT C A A A A T TC C G

T T C missing

missing

Stop

53

35

35

Met Phe Gly

3 nucleotide-pair deletion

A GU C A AG GU U U U

T GA A AT T TT CG G

A A G

Accuracy

• Transcription less accurate than replication– Errors occur 1 in 10000 nucleotides

• Protein synthesis more tolerant of errors than replication– Redundancy of codons for amino acids

Documents

From Gene to Protein Chapter 17. What Is a Gene? The idea of the gene has evolved through the history of genetics We have considered a gene as – A discrete