1

TRANSCRIPTION RNA is transcribedfrom a DNA template.

DNA

RNApolymerase

RNAtranscript

RNA PROCESSING In eukaryotes, theRNA transcript (pre-mRNA) is spliced andmodified to producemRNA, which movesfrom the nucleus to thecytoplasm.

Exon

Poly-A

RNA transcript(pre-mRNA)

Intron

NUCLEUSCap

FORMATION OFINITIATION COMPLEX

After leaving thenucleus, mRNA attachesto the ribosome.

CYTOPLASM

mRNA

Poly-A

Growingpolypeptide

Ribosomalsubunits

Cap

Aminoacyl-tRNAsynthetase

AminoacidtRNA

AMINO ACID ACTIVATION

Each amino acidattaches to its proper tRNAwith the help of a specificenzyme and ATP.

Activatedamino acid

TRANSLATION A succession of tRNAsadd their amino acids tothe polypeptide chainas the mRNA is movedthrough the ribosomeone codon at a time.(When completed, thepolypeptide is releasedfrom the ribosome.)

Anticodon

A CC

A A AUGGUU UA UG

U ACE A

Ribosome

1

Poly-A

5′

5′

3′

Codon

2

3 4

5

From Gene to Phenotype- part 3DNA

mRNA

polypeptide

Lecture Outline 11/9/05• Review translation:

– Initiation, elongation, termination– EPA model

• Post-translational modification of polypeptides• Signal sequences• Mutations (again)

Exam 3 is next Monday. It will cover mitosis andmeiosis, DNA synthesis, transcription, translation,genetics of viruses.(chapters 12, 13, 16, 17, part of 18 (to page 345))

Translation: overviewTRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

Polypeptide

Aminoacids

tRNA withamino acidattachedRibosome

tRNA

Anticodon

mRNA

Trp

Phe Gly

A G C

A A A

CC

G

U G G U U U G G C

Codons5′ 3′

The ribosome is themachine that builds thepolypeptide

tRNA serves asan “adaptor”that brings thecorrect aminoacid to eachcodon.

The genetic codeSecond mRNA base

U C A G

U

C

A

G

UUU

UUCUUAUUG

CUUCUCCUACUG

AUUAUCAUAAUG

GUUGUCGUAGUG

Met orstart

Phe

Leu

Leu

lle

Val

UCU

UCCUCAUCG

CCUCCCCCACCG

ACUACCACAACG

GCUGCCGCAGCG

Ser

Pro

Thr

Ala

UAUUAC

UGU

UGCTyr Cys

CAUCACCAACAG

CGUCGCCGACGG

AAUAACAAAAAG

AGU

AGCAGAAGG

GAUGACGAAGAG

GGUGGCGGAGGG

UGG

UAAUAG Stop

Stop UGA Stop

Trp

His

Gln

Asn

Lys

Asp

Arg

Ser

Arg

Gly

U

CA

GUCAG

UCAG

UCAG

Firs

t mR

NA

bas

e (5′ e

nd)

Third

mR

NA

bas

e (3′ e

nd)

Glu

U

C

A

G

U C A GUCAGUCAGUCAGUCAG

3′ACCACGCUUAAGACACCU

GC *

GU GUCU

GAGGU

A

A A GUC

AGACC

CGAGAG GG

GACUCAUUUAGGCG5′

Hydrogenbonds

*

*

**

*

**

*

* **

5’-AUGCAAUUCGGAAAC

Codon in the mRNA

2

4

An aminoacyl-tRNA synthetase joins aspecific amino acid to a tRNA

Amino acid

ATP

Adenosine

Pyrophosphate

Adenosine

Adenosine

tRNA

P P P

P

P Pi

PiPi

P

AMP

AppropriatetRNA bonds to amino

Acid, displacingAMP.

Active site binds theamino acid and ATP. 1

3

Aminoacyl-tRNAsynthetase (enzyme)

Activated amino acidis released by the enzyme.

Each tRNA has aslightly differentshape

How does the ribosome findAUG?

• Prokaryotes have a special bindingsequence upstream of the start codon.

• In Eukaryotes,the ribosome binds to the5’ cap and “scans” the message for anAUG.

See the Animation

• www.dnai.org

Inhibition of protein synthesisToxin Mode of action Target

Puromycinforms peptidyl-puromycin, prevents

translocationProcaryotes

Tetracyclineblocks the A-site, prevents binding of aminoacyl

tRNAsProcaryotes

Chloramphenicol blocks peptidyl transfer Procaryotes

Cycloheximide blocks peptidyl transferase Eucaryotes

Streptomycin inhibits initiation at high concentrations Procaryotes

Diphtheria toxin catalyzes ADP-ribosylation of residue in eEF2 Eucaryotes

Erythromycin binds to 50S subunit, inhibits translocation Procaryotes

Ricininactivates 60S subunit, depurinates an

adenosine in 23S rRNAEucaryotes

NOTE: Prokaryotes (this generally includes proteinsynthesis in mitochondria and chloroplasts)

3

Only the anticodon of tRNA determineswhich amino acid is added by a

ribosome.

• Experimental evidence:– Convert cystein to alanine chemically, after

it is attached to tRNA (remove SH group)– Alanine shows up in Cystein sites

The amino acid carried by a tRNA isindependent of the anticodon sequence

• Determined by the amino-acyl tRNAsynthetase enzyme– tRNA with mutations in the anticodon still

have their normal amino acid at the 3’end.

– Experiment:. mutate anticodon of tRNAthr

(AGU-->AGG)• Now binds to proline codon instead (CCU).• Those tRNA still carry threonine, but now

bind to proline sites.• Threonine inserted into polypeptide where

proline normally goes.

Glycine doesn’t fit . .

Alananine tRNA synthetaseAminoacyl tRNAsynthetaseenzyme isspecific to aparticular aminoacid and aparticular tRNA

Quality control

• Both cap and tail bind to initiation factors tostart translation– Ensures that mRNA is intact

• Small subunit can detect mis-paired tRNAand remove them– Needs a short delay before peptide bond is formed

(to give time for proofreading)

• Error rate: about 10-4

4

Cost of protein biosynthesis

• Synthesis of aminoacyl tRNAs 2 ATPs• Formation of 1 peptide bond 2 GTPs

– 1 for codon recognition; 1 for translocation

• Proofreading 1 ATP/error

• Construction of a specific amino acid sequence ismuch more costly than formation of a randompeptide bond!

Transcription and translation canoccur simultaneously

DNA

Polyribosome

mRNA

Direction oftranscription

0.25 µmRNApolymerase

Ribosome

DNA

mRNA (5′ end)

RNA polymerase

Polypeptide

Post translationalmodifications and sorting

Glycosylation

Signal directs protein to the right compartment

The signal mechanism for targetingproteins to the ER

Foldsto finalshape

Translationbegins inthe cytosol

SRP binds to the signal peptide,

Attaches totranslocationpore in ERmembrane

Polypeptidesynthesizedinto the ER

Signalpeptideremoved

1 2 3 4 5 6

Ribosome

mRNASignalpeptide

Signal-recognitionparticle(SRP) SRP

receptorprotein

Translocationcomplex

CYTOSOL

Signalpeptideremoved

ERmembrane

Protein

5

Brooker Figure 13.22

Destined for ERDestined for cytosolor other organelles

Importedduringtranslation

Importedaftertranslation

Signal peptidedetermines where itgoes

Stays within themembranesystem

Chaperones help fold proteinsHsp 70 covers exposedhydrophobic patches untilthe protein can fold

Hsp60 is like an isolation chamber

Mis-folded proteins aremarked for destruction with

ubiquitinUbiquitin tail

Proteosome acts as garbage disposal

6

Mutations (again)

The molecular basis of sickle-celldisease: a point mutation

In the DNA, themutant templatestrand has an A where the wild-type template has a T.

The mutant mRNA has a U instead of an A in one codon.

The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu).

Mutant hemoglobin DNAWild-type hemoglobin DNA

mRNA mRNA

Normal hemoglobin Sickle-cell hemoglobin

Glu Val

C T T C A T

G A A G U A

3′ 5′ 3′ 5′

5′ 3′5′ 3′

Base-pair substitutionWild type

A U G A A G U U U G G C U A AmRNA 5′Protein Met Lys Phe Gly Stop

Carboxyl endAmino end

3′

A U G A A G U U U G G U U A A

Met Lys Phe Gly

Base-pair substitutionNo effect on amino acid sequence

U instead of C

Stop

A U G A A G U U U A G U U A A

Met Lys Phe Ser Stop

A U G U A G U U U G G C U A A

Met Stop

Missense A instead of G

NonsenseU instead of A

Base-pair insertion or deletionmRNAProtein

Wild type

A U G A A G U U U G G C U A A5′

Met Lys Phe Gly

Amino end Carboxyl end

Stop

Base-pair insertion or deletionFrameshift causing immediate nonsense

A U G U A A G U U U G G C U A

A U G A A G U U G G C U A A

A U G U U U G G C U A A

Met Stop

U

Met Lys Leu Ala

Met Phe GlyStop

MissingA A G

Missing

Extra U

Frameshift causing extensive missense

Insertion or deletion of 3 nucleotides:no frameshift but extra or missing amino acid

3′

7

Mutations in the 3rd positionof a codon are often silent

Second mRNA baseU C A G

U

C

A

G

UUUUUCUUAUUG

CUUCUCCUACUG

AUUAUCAUAAUG

GUUGUCGUAGUG

Met orstart

Phe

Leu

Leu

lle

Val

UCUUCCUCAUCG

CCUCCCCCACCG

ACUACCACAACG

GCUGCCGCAGCG

Ser

Pro

Thr

Ala

UAU

UACUGUUGC

Tyr Cys

CAUCACCAACAG

CGUCGCCGACGG

AAUAACAAAAAG

AGU

AGCAGAAGG

GAUGACGAAGAG

GGUGGCGGAGGG

UGG

UAAUAG Stop

Stop UGA Stop

Trp

His

Gln

Asn

Lys

Asp

Arg

Ser

Arg

Gly

U

CA

GUCAG

UCAG

UCAG

Firs

t mR

NA

bas

e (5′ e

nd)

Third

mR

NA

bas

e (3′ e

nd)

Glu

For amino acids thathave only two codons,the 3rd base will eitherboth be purines or bothbe pyrimidines

Wobble in 3rd position