chapter 17: from gene to protein 17.pdfthe mrna transcript is: 5’-caugccugggcaauag-3’ the...

Chapter 17:

From Gene to Protein

4. Translation

3. The Genetic Code

2. Transcription

1. Overview of Gene Expression

5. Mutations

1. Overview of Gene Expression

Chapter Reading – pp. 334-337

How are Genes related to DNA?

Genes are segments of DNA that code for a

particular protein (or RNA molecule)

• the human genome contains ~3 billion base

pairs (bps) and ~25,000 genes

• most genes encode proteins

• when we talk about “genes” we will focus on those

that express proteins

• the gene products of some genes are RNA

molecules that play a variety of roles in cells

Minimal medium

No growth:

Mutant cells

cannot grow

and divide

Growth:

Wild-type

cells growing

and dividing

EXPERIMENT RESULTS

CONCLUSION

Classes of Neurospora crassa

Wild type Class I mutants Class II mutants Class III mutants

Minimalmedium(MM)(control)

MM ornithine

MM citrulline

Co

nd

itio

n

MM arginine(control)

Summaryof results

Can grow withor without anysupplements

Can grow onornithine,citrulline, orarginine

Can grow onlyon citrulline orarginine

Require arginineto grow

Wild type

Class I mutants

(mutation in

gene A)

Class II mutants

(mutation in

gene B)

Class III mutants

(mutation in

gene C)

Gene

(codes for

enzyme)

Gene A

Gene B

Gene C

Precursor Precursor Precursor Precursor

Enzyme A Enzyme A Enzyme A Enzyme A

Enzyme B Enzyme B Enzyme B Enzyme B

Enzyme C Enzyme C Enzyme C Enzyme C

Ornithine Ornithine Ornithine Ornithine

Citrulline Citrulline Citrulline Citrulline

Arginine Arginine Arginine Arginine

• these classic

experiments led to

the “one gene-

one enzyme”

hypothesis

• we now know that

genes code for

more than just

enzymes

Beadle & Tatum, 1941

Srb & Horowitz, 1944

Gene Expression

The expression of most genes involves two

distinct processes:

1) Transcription of a gene into RNA

• this is essentially creating a “photocopy” of the gene

• occurs in the nucleus

2) Translation of the RNA transcript into protein

• accomplished by ribosomes, in the cytoplasm

• RNA is a nucleic acid very similar to DNA

(RNA uses “U” instead of “T”)

DNA

mRNA

Ribosome

Polypeptide

TRANSCRIPTION

TRANSLATION

TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome

DNA

mRNA

Pre-mRNARNA PROCESSING

(a) Bacterial cell (b) Eukaryotic cell

Nuclear

envelope

DNA RNA Protein

Prokaryotes vs Eukaryotes

DNAtemplatestrand

TRANSCRIPTION

mRNA

TRANSLATION

Protein

Amino acid

Codon

Trp Phe Gly

5

5

Ser

U U U U U

3

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 G

DNA

molecule

Gene 1

Gene 2

Gene 3

C C

From DNA to RNA to Protein

• gene expression ends at transcription for genes encoding

RNA gene products

Comparison of DNA & RNA

• sugar = Ribose

• single-stranded

• A, C, G & U (uracil)

RNA DNA

• sugar = Deoxyribose

• double-stranded

• A, C, G & T (thymine)

2. Transcription


Stages of

Transcription

INITIATIONRNA polymerase binds

to the promoter of a

gene, separates DNA

ELONGATIONRNA polymerase makes

RNA complementary to

the template strand

TERMINATIONRNA polymerase stops

transcribing when end of

gene is reached

Promoter

RNA polymerase

Start pointDNA

53

Transcription unit

35

Elongation

53

35

Nontemplate strand of DNA

Template strand of DNARNAtranscript

UnwoundDNA

2

353

53

RewoundDNA

RNAtranscript

5

Termination3

3

5

5Completed RNA transcript

Direction of transcription (“downstream”)

53

3

Initiation1

Transcription initiation

complex forms

3

DNA

PromoterNontemplate strand

53

53

53

Transcription

factors

RNA polymerase II

Transcription factors

53

5

3

53

RNA transcript

Transcription initiation complex

53

TATA box

T

T T T T T

A A A AA

A A

T

Several transcription

factors bind to DNA

2

A eukaryotic promoter1

Start point Template strand

Initiation of

Eukaryotic

Transcription

Eukaryotic promoters

contain a short

sequence called a

TATA box.

With the help of an

array of transcription

factors, RNA

polymerase binds to

the promoter and

starts transcription.

Nontemplate

strand of DNA

RNA nucleotides

RNA

polymerase

Template

strand of DNA

3

35

5

5

3

Newly made

RNA

Direction of transcription

A A A

A

T

TTT G

C

C C

G

C CC A AU

end

ELONGATION

In self-termination, the transcription of DNA

terminator sequences cause the RNA to fold,

loosening the grip of RNA polymerase on the DNA.

In enzyme-dependent termination, a termination

enzyme pushes between RNA polymerase and

the DNA, releasing the polymerase.

Termination of transcription

RNA transcript released

RNA polymerase

released

Rho termination

protein

3

5

Rho protein moves

along RNA

3

C-G rich

stem-loop

Termination of Transcription

• triggered by stem/loop structure in RNA or termination

factors such as the Rho protein (prokaryotes)

Protein-coding

segmentPolyadenylation

signal5 3

35 5Cap UTRStart

codon

G P P P

Stop

codon UTR

AAUAAA

Poly-A tail

AAA AAA…

Primary RNA TranscriptsNewly made RNA molecules in eukaryotes are called

primary transcripts because they need to be

processed before they can carry out their function:

mRNA 1o transcript

• a 5’ cap

Messenger (mRNA) primary transcripts require the

following modifications:

• a poly-A tail

• splicing out of introns

5 Exon Intron Exon

5 CapPre-mRNACodonnumbers

130 31104

mRNA 5 Cap

5

Intron Exon

3 UTR

Introns cut out and

exons spliced together

3

105

146

Poly-A tail

Coding

segment

Poly-A tail

UTR1146

RNA Splicing

The coding region of the primary RNA transcript

contains intervening sequences called introns that

need to be removed or “spliced out”.

The regions that are retained are called exons which

after splicing form a continuous coding region.

RNA transcript (pre-mRNA)5

Exon 1

Protein

snRNA

snRNPs

Intron Exon 2

Other proteins

Spliceosome

5

Spliceosome

componentsCut-out

intronmRNA5

Exon 1 Exon 2

How is

Splicing

Carried

Out?

Spliceosomes are

structures made of

protein and snRNA

snRNA in

spliceosome base

pairs with ends of

intron sequences

resulting in their

being “spliced out”

Exons and Protein Structure

Exons tend to

code for distinct

substructures

called domains

in proteins.

Exon shuffling is

thought to be an

evolutionary

process by

which new

genes are made.

GeneDNA

Exon 1 Exon 2 Exon 3Intron Intron

Transcription

RNA processing

Translation

Domain 3

Domain 2

Domain 1

Polypeptide

Various Roles of RNA Transcripts

1) messenger RNA (mRNA)

• RNA copy of a gene that encodes a polypeptide

2) ribosomal RNA (rRNA)

• RNA that is a structural component of ribosomes

3) transfer RNA (tRNA)

• delivery of “correct”

amino acids to

ribosomes during

translation

For some genes, the end-product

is the RNA itself (rRNA, tRNA)

3. The Genetic Code


How do Genes code for Proteins?

Recall that proteins are linear polymers

made of 20 different amino acids.

Genes need simply to encode the identity of

each amino acid in a given protein!

• i.e., genes must be capable of encoding

20 different amino acids and their order in a

protein

• although DNA contains only 4 different

nucleotides, this is more than sufficient to

specify 20 different amino acids…

Basis of the Genetic Code

Each amino acid in a protein is specified by

3 nucleotide sequences called codons

• there are 64 possible “codon” triplets (4 x 4 x 4)

• more than enough to encode 20 amino acids and the

signal to “stop” or end the protein (TGA, TAA or TAG)

• each of the 20 amino acids is coded for by a

unique set of codons:

e.g. ATG = methionine (start codon)

GGN = glycine

CAA or CAG = glutamine

If the DNA sequence is:5’-CATGCCTGGGCAATAG-3’

3’-GTACGGACCCGTTATC-5’

The mRNA transcript is: 5’-CAUGCCUGGGCAAUAG-3’

The polypeptide is:

*Met-Pro-Gly-Gln (stop)

all proteins begin w/Met

(transcription)

(translation)

The Genetic CodeSecond mRNA base

Fir

st

mR

NA

base (

5en

d o

f co

do

n)

Th

ird

mR

NA

base (

3en

d o

f co

do

n)

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 or

start

UUG

G

(a) Tobacco plant expressing a firefly gene gene

(b) Pig expressing a jellyfish

The Genetic Code is UniversalGenes from one species can be expressed in another!

4. Translation

Chapter Reading – pp. 83, 345-354

Polypeptide

Ribosome

tRNA with

amino acid

attached

Amino

acids

tRNA

Anticodon

Codons

U U U UG G G G C

C

G

C

G

5 3

mRNA

Overview of

Translation

Ribosomes facilitate

the production of

polypeptides by:

1) matching codons

in mRNA with

complementary

anticodons in tRNA

2) catalyzing peptide

bonds between

amino acids carried

by tRNAs

tRNA

molecules

Growing

polypeptide Exit tunnel

E PA

Large

subunit

Small

subunit

mRNA5

3

(a) Computer model of functioning ribosome

Exit tunnel Amino end

A site (Aminoacyl-

tRNA binding site)

Small

subunit

Large

subunit

E P AmRNA

E

P site (Peptidyl-tRNA

binding site)

mRNA

binding site

(b) Schematic model showing binding sites

E site

(Exit site)

(c) Schematic model with mRNA and tRNA

5 Codons

3

tRNA

Growing polypeptide

Next amino

acid to be

added to

polypeptide

chain

Ribosome

Structure

Amino acid

attachment

site

3

5

Hydrogen

bonds

Anticodon

(a) Two-dimensional structure (b) Three-dimensional structure

(c) Symbol used in this book

Anticodon Anticodon

3 5

Hydrogen

bonds

Amino acid

attachment

site5

3

A A G

Transfer RNA (tRNA)tRNA anticodon

will base pair with

a complementary

codon in mRNA

Aminoacyl-tRNA

synthetase (enzyme)

Amino acid

P P P Adenosine

ATP

P

P

P

PPi

i

i

Adenosine

tRNA

AdenosineP

tRNA

AMP

Computer model

Amino

acid

Aminoacyl-tRNA

synthetase

Aminoacyl tRNA

(“charged tRNA”)

Aminoacyl-tRNA

Synthetases

Each tRNA

is loaded

with the

correct

amino acid

due to the

action of

these

enzymes.

Initiator

tRNA

mRNA

5

53

Start codonSmall

ribosomal

subunitmRNA binding site

3

Translation initiation complex

5 3

3 U

U

AA G

C

P

P site

i

GTP GDP

Large

ribosomal

subunit

E A

5

Initiation of Translation

• small ribosomal subunit aligns with start codon of mRNA

• initiator tRNAmet and large subunit then join the complex

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

Elongation

Cycle of

Translation

Releasefactor

Stop codon

(UAG, UAA, or UGA)

3

5

3

5

Freepolypeptide

2 GTP

5

3

2 GDP 2i

P

Termination of Translation

When a stop codon is reached there is no tRNA

with a complementary anticodon.

Instead a release factor fits in the A site and

catalyzes the dissociation of all components.

Multiple

Ribosomes

translate the

same mRNA

Referred to as

polyribosomes.

Completed

polypeptide

Incoming

ribosomal

subunits

Start of

mRNA

(5 end)

End of

mRNA

(3 end)(a)

Ribosomes

mRNA

(b)0.1 m

Growing

polypeptides

The cap comesoff, and the properlyfolded protein isreleased.


Hollowcylinder

Cap

Chaperonin(fully assembled)

Polypeptide

Steps of ChaperoninAction:

An unfolded poly-peptide enters thecylinder from one end.

1

2 3The cap attaches, causing thecylinder to change shape insuch a way that it creates ahydrophilic environment forthe folding of the polypeptide.


Correctlyfoldedprotein

Ensuring Proper Protein Structure (pg. 83)

Chaperonins are large protein structures that assist

newly made polypeptides to ensure they fold properly.

• capture improperly folded proteins inside its cavity, forcing

them to unfold and hopefully refold correctly

Ribosome

mRNA

Signalpeptide

SRP

1

SRPreceptorprotein

Translocationcomplex

ERLUMEN

2

3

45

6

Signalpeptideremoved

CYTOSOL

Protein

ERmembrane

Translation in the ER

Proteins destined for the “secretory pathway”

have a signal peptide at the N-terminus that

targets the protein to the ER lumen.

Gene Expression in Prokaryotes

Transcription and

translation are

not segregated in

prokaryotes.

Since prokaryotic

RNA transcripts

don’t need to be

processed,

translation can

begin before

transcription is

finished!

RNA polymerase

DNAmRNA

Polyribosome

RNA

polymeraseDNA

Polyribosome

Polypeptide

(amino end)

mRNA (5 end)

Ribosome

0.25 mDirection of

transcription

TRANSCRIPTIONDNA

RNApolymerase

Exon

RNAtranscript

RNAPROCESSING

NUCLEUS

Intron

RNA transcript(pre-mRNA)

Aminoacyl-tRNA synthetase

AMINO ACIDACTIVATION

Aminoacid

tRNA

3

GrowingpolypeptidemRNA

Aminoacyl(charged)tRNA

Anticodon

Ribosomalsubunits

A

AE

TRANSLATION

CYTOPLASM

P

E

Codon

Ribosome

5

3

Summary

of Gene

Expression

5. Mutation


Mutations

A mutation is any change in DNA sequence:

• change of one nucleotide to another

• insertion or deletion of nucleotides or DNA fragments

• inversion or recombination of DNA fragments

What causes mutations?

• errors in DNA replication or DNA repair

• chemical mutagenesis

• high energy electromagnetic radiation

• UV light, X-rays, gamma rays

Sickle-cell Anemia

• a single nucleotide change causes a single amino

acid change resulting in a malformed protein

Wild-type hemoglobin

Wild-type hemoglobin DNA

3

3

35

5 3

35

5

553

mRNA

A AG

C T T

A AG

mRNA

Normal hemoglobin

Glu

Sickle-cell hemoglobin

Val

A

A

AUG

G

T

T

Sickle-cell hemoglobin

Mutant hemoglobin DNA

C

Types of Mutations

Silent mutations:

• have no effect on amino acid specification

Missense mutations:

• result in the change of a single amino acid

Nonsense mutations:

• convert a codon specifying an amino acid to a stop codon

• results in premature truncation of a protein

Insertion/deletion mutations:

• cause a shift in the reading frame of the gene

• all codons downstream of insertion/deletion will be incorrect

Silent MutationsWild type

DNA template strand

mRNA5

5

Protein

Amino end

Stop

Carboxyl end

3

3

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

5

3

3

5A

A A

A A A A

A AT

T T T T T

T T TT

C C C C

G G G G

A

A

A G A A A AG GGU U U U U

T

U 35

Missense MutationsWild type

DNA template strand

mRNA5

5

Protein

Amino end

Stop

Carboxyl end

3

3

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


5

3

3

5A

A A

A A A A

A AT

T T T T T

T T TT

C C T C

G

G

GA

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

A C

C

G

Nonsense MutationsWild type

DNA template strand

mRNA5

5

Protein

Amino end

Stop

Carboxyl end

3

3

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


5

3

3

5A

A

A A A A

A AT

T A T T T

T T TT

C 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 A

G

Stop

Wild type

DNA template strand

mRNA5

5

Protein

Amino end

Stop

Carboxyl end

3

3

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 causing

immediate nonsense

Extra A

Extra U

5

3

5

3

3

5

Met

1 nucleotide-pair insertion

Stop

A C A A GT T A TC T A C G

T A T AT G T CT GG A T GA

A G U A U AU GAU G U U C

A T

A

AG

Frameshift – Insertion/Deletion

Can cause a premature

stop codon…

DNA template strand

mRNA5

5

Protein

Amino endStop

Carboxyl end

3

3

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 causing

extensive 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

5

3

3

5

Met Lys Leu Ala

1 nucleotide-pair deletion

5


…or

extended

shift in

reading

frame…

DNA template strand

mRNA5

5

Protein

Amino end

Stop

Carboxyl end

3

3

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 one

amino acid missing

Wild type

AT C A A A A T TC C G

T T C missing

missing

Stop

5

3

3

5

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


…or the

addition/

deletion of

a single

amino acid.

Must be a

multiple

of 3!

Key Terms for Chapter 17

• transcription, promoter, TATA box, RNA polymerase

• primary transcript, mRNA, tRNA, rRNA, snRNA

• 5’ cap, poly-A tail, intron, exon, splicing, spliceosome

• rho protein, stem-loop, codon, anti-codon, translation

• mutation: substitution, deletion, insertion

• silent, missense, nonsense mutations

• aminoacyl tRNA synthetase, polyribosome, signal

peptide, release factor, chaperonin

• reading frame, frameshift

Relevant

Chapter

Questions 1-9, 11

chapter 17: from gene to protein 17.pdfthe mrna transcript is: 5’-caugccugggcaauag-3’ the...

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