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 AUG GUU UA U G

UACE A

Ribosome

1

Poly-A

5

5

3

Codon

2

3 4

5

From Gene to Phenotype- part 3

DNA

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 and meiosis, 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 acidattached

Ribosome

tRNA

Anticodon

mRNA

Trp

Phe Gly

AG C

A A A

CC

G

U G G U U U G G C

Codons5 3

The ribosome is the machine that builds the polypeptide

tRNA serves as an “adaptor” that brings the correct amino acid to each codon.

The genetic codeSecond mRNA base

U C A G

U

C

A

G

UUU

UUCUUA

UUG

CUU

CUC

CUA

CUG

AUU

AUC

AUA

AUG

GUU

GUC

GUA

GUG

Met orstart

Phe

Leu

Leu

lle

Val

UCU

UCCUCA

UCG

CCU

CCC

CCA

CCG

ACU

ACC

ACA

ACG

GCU

GCC

GCA

GCG

Ser

Pro

Thr

Ala

UAU

UAC

UGU

UGCTyr Cys

CAU

CAC

CAA

CAG

CGU

CGC

CGA

CGG

AAU

AAC

AAA

AAG

AGU

AGC

AGA

AGG

GAU

GAC

GAA

GAG

GGU

GGC

GGA

GGG

UGG

UAA

UAG Stop

Stop UGA Stop

Trp

His

Gln

Asn

Lys

Asp

Arg

Ser

Arg

Gly

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

Fir

st m

RN

A b

ase

(5

end

)

Th

ird

mR

NA

ba

se (

3 e

nd

)Glu

U

C

A

G

U C A GUCAG

UCAG

UCAG

UCAG

3ACCACGCUUAA

GACACCU

GC

*GU GU

CUGAG

GU

A

AA G

UC

AGACC

CGA GA G G

G

GACUCAU

UUAGGCG5

Hydrogenbonds

*

*

**

*

**

*

* **

5’-AUGCAAUUCGGAAAC

Codon in the mRNA

4

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

Amino acid

ATP

Adenosine

Pyrophosphate

Adenosine

Adenosine

tRNA

P P P

P

P Pi

Pi

Pi

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 a slightly different shape

How does the ribosome find AUG?

• Prokaryotes have a special binding sequence upstream of the start codon.

• In Eukaryotes,the ribosome binds to the 5’ cap and “scans” the message for an AUG.

See the Animation

• www.dnai.org

Inhibition of protein synthesisToxin Mode of action Target

Puromycinforms peptidyl-puromycin, prevents translocation

Procaryotes

Tetracyclineblocks the A-site, prevents binding of aminoacyl tRNAs

Procaryotes

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 rRNA

Eucaryotes

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

Only the anticodon of tRNA determines which 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 is independent of the anticodon sequence

• Determined by the amino-acyl tRNA synthetase 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 synthetase

Aminoacyl tRNA synthetase enzyme is specific to a particular amino acid and a particular tRNA

Quality control

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

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

(to give time for proofreading)

• Error rate: about 10-4

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 is much more costly than formation of a random peptide bond!

Transcription and translation can occur simultaneously

DNA

Polyribosome

mRNA

Direction oftranscription

0.25 mRNApolymerase

Ribosome

DNA

mRNA (5 end)

RNA polymerase

Polypeptide

Post translational modifications and sorting

Glycosylation

Signal directs protein to the right compartment

The signal mechanism for targeting proteins to the ER

Folds to final shape

Translation begins in the cytosol

SRP binds to the signal peptide,

Attaches to translocation pore in ER membrane

Polypeptide synthesized into the ER

Signal peptide removed

1 2 3 4 5 6

Ribosome

mRNASignalpeptide

Signal-recognitionparticle(SRP) SRP

receptorprotein

Translocationcomplex

CYTOSOL

Signalpeptideremoved

ERmembrane

Protein

Brooker Figure 13.22

Destined for ERDestined for cytosol or other organelles

Imported during translation

Imported after translation

Signal peptide determines where it goes

Stays within the membrane system

Chaperones help fold proteinsHsp 70 covers exposed hydrophobic patches until the protein can fold

Hsp60 is like an isolation chamber

Mis-folded proteins are marked for destruction with

ubiquitinUbiquitin tail

Proteosome acts as garbage disposal

Mutations (again)

The molecular basis of sickle-cell disease: 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 35 3

Base-pair substitutionWild type

A U G A A G U U U G G C U A AmRNA5

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 substitution

No effect on amino acid sequenceU 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 deletion

mRNA

Protein

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 deletion

Frameshift 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

Mutations in the 3rd position of a codon are often silent

Second mRNA base

U C A G

U

C

A

G

UUU

UUCUUA

UUG

CUU

CUC

CUA

CUG

AUU

AUC

AUA

AUG

GUU

GUC

GUA

GUG

Met orstart

Phe

Leu

Leu

lle

Val

UCU

UCCUCA

UCG

CCU

CCC

CCA

CCG

ACU

ACC

ACA

ACG

GCU

GCC

GCA

GCG

Ser

Pro

Thr

Ala

UAU

UAC

UGU

UGCTyr Cys

CAU

CAC

CAA

CAG

CGU

CGC

CGA

CGG

AAU

AAC

AAA

AAG

AGU

AGC

AGA

AGG

GAU

GAC

GAA

GAG

GGU

GGC

GGA

GGG

UGG

UAA

UAG Stop

Stop UGA Stop

Trp

His

Gln

Asn

Lys

Asp

Arg

Ser

Arg

Gly

U

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

Fir

st m

RN

A b

ase

(5

end

)

Th

ird

mR

NA

bas

e (3

en

d)

Glu

For amino acids that have only two codons, the 3rd base will either both be purines or both be pyrimidines

Wobble in 3rd position