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Translation . Introduction 1 -the genetic code 2 -tRNAs codon recognition and aminoacylation 3 -Translation initiation 4 -Translation elongation 5-Peptide bond formation catalysis in the ribosome 6-Translation termination 7-Quality Control of translation. Fig 30-7 Voet and Voet :

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slide1

Translation

Introduction

1-the genetic code

2-tRNAs codon recognition

and aminoacylation

3-Translation initiation

4-Translation elongation

5-Peptide bond formation

catalysis in the ribosome

6-Translation termination

7-Quality Control of translation

Fig 30-7 Voet and Voet:

What’s wrong with that picture ?

Not treated: Translation Regulation

(initiation, elongation, termination)

slide2

The

Genetic

Code

slide3

tRNAs are adaptor molecules

Crick’s

Adaptor

Hypothesis

slide4

One tRNA can recognize

several codons

AntiCodonCodon

(tRNA) (mRNA)

C G

A U

U A,G

G C,U

I C,U,A

slide5

Biological Peptide Formation is

NOT a condensation reaction

Biological

Peptide Bond

Formation

Condensation

slide6

Aminoacylation

of tRNAs

Interests:

1)Provide the adaptor for translation

2)Activates the carboxyl group

of the amino acid

for peptide bond formation

slide7

Fidelity of tRNA Amino acylation

- Discrimination of the “right” AA on a single step can not exceed 1/100

- AA tRNAsynthetases specificity occurs by two steps

1)Charging of the amino acid on the RNA in the “synthetic

Domain”

2) Shuttling of

the charged

amino acyl-tRNA

in the editing

site

Correct Charging

This would result

in errors

Incorrect

Charging

Reynolds et al.

Nat.Rev.Microbiol.2010

slide8

A functional comparison

between DNA Polymerase

and tRNAsynthetases

Synthetic mode --> Editing mode

Synthetic mode --> Editing mode

slide9

Prokaryotes

Eukaryotes

50S

60S

2820 kDa

1590 kDa

Large

Subunit

28S, 5S, 5.8 S

rRNAs

23S, 5S

rRNAs

Ribosomes

Composition

49 proteins

31 proteins

Peptidyl Transferase

Center

Peptidyl Transferase

Center

30S

40S

930 kDa

1400 kDa

Small

subunit

16S rRNA

18 S rRNA

21 proteins

33 proteins

mRNA Binding

Error Correction

mRNA Binding

Error Correction

slide10

mRNA binding

Error correction

70S ribosome functions

Small Subunit

Large Subunit

Peptidyl transferase

Cates et al., Science (1999)

slide11

Prokaryotic translation initiation

Translation

immediately

follows

transcription

in Prokaryotes

Translation of a polycistronic

mRNA in prokaryotes

ORF1

ORF2

ORF3

Stop

AUG

SD

Translation

stops

Translation

starts

Ribosome

Dissociates

Ribosome

Binds to

Shine-Dalgarno

sequence

slide12

Translation

immediately

follows

transcription

in Prokaryotes

From the relative sizes of the nascent polypeptides, can you pinpoint the direction of translation and the 5’-3’ polarity of the mRNA on this electron micrograph ?

slide13

Eukaryotic translation initiation

1) Binding of the

mRNA by the 40S

subunit+initiation

factors

2) Scanning of the

mRNA to search

for AUG

3) Binding of the

60S subunit

slide14

Recognition of the 5’-cap structure of eukaryotic mRNAs by eIF4e

Cap binding pocket is on the concave

surface of the protein – allows easy

entry of the mRNA 5’-end

PDB ID = 1L8B

Niedzwiecka et al. J.Mol.Biol. 2002

• base sandwich-stacking between Trp56 and Trp102

• formation of three Watson–Crick-like hydrogen bonds with

Glu103 and backbone NH of Trp102

= H20

• van der Waals contact of the N(7)-methyl group with Trp166

slide15

Simplified view of the elongation and termination reactions in the ribosome

Leung et al. Ann.Rev. Biochem. 2010

slide16

Ribosome Recycling Factor (RRF)

resembles a tRNA molecule

A case of molecular mimickry !

This allows RRF to

enter the empty A site dyring

translation termination

Blue =

RRF

Red = yeast

tRNAPhe

PDB ID = 1DD5

Selmer et al. Science 1999:Vol. 286. no. 5448, pp. 2349 - 2352

slide17

Translation Elongation in Prokaryotes:

2 independentFidelity Mechanisms

Normal Elongation(codons and tRNAs are matched –see color code)

Aberrant Elongation

Incorporation of an incorrect amino

acids promotes an even greater loss of fidelity

for the next round of elongation, and ultimately

triggers peptide release and dissociation

Reynolds et al.

Nat.Rev.Microbiol.2010

slide18

Peptide Bond Formation:

The Peptidyl

Transferase

Reaction

slide19

Evidences for RNA Catalysis in the Peptidyltransferase center

Biochemical Evidence:

SubunitCatalytic Efficiency

50S E.coli subunit 100%

Deproteinized 50S E.coli 20%

50S T.aquaticus 100%

Deproteinized 50S T.aquaticus 80%

Deproteinized 50S T.aquaticus+RNAse 0%

Deproteinized = 95% of the proteins are removed

Structural Evidence:

•There is no protein with >10 angstrom of the

catalytic center of the peptidyltransferase

reaction in the 50S subunit

---> The reaction has to be catalyzed by RNA

---> 1 Adenine base is perfectly localized for this.

Yellow = Proteins

Grey = RNA Green = Peptidyl Transferase Center

slide20

Proposed Catalysis Model in the Peptidyltransferase center

T.Steitz - Nature Reviews Mol.Cell.Biol.

Vol.9 - March 2008

Proposed Catalysis Model :

• crucial role of the 2’hydroxyl of A76

of the tRNAas a proton Shuttle” in receiving a proton from the  amino group (A site - labeled “N”) and transferring it to the 3’O

of the tRNA-peptide link in the P site

Leung et al. Ann.Rev. Biochem. 2010

slide21

Quality Control of Translation:

These quality control pathways prevent the accumulation of

truncated or deficient proteins due to defects in mRNAs:

- truncated mRNAs (because of random breaks in RNAs)

- mRNAs that do not contain stop codons (because of mistakes

in the 3’-end processing reactions)

- mRNAs containing premature termination codons (gene

mutations or errors made by RNA polymerase)

2 examples of Translation QC in eukaryotes

• “Nonstop” protein degradation in eukaryotes

•  Nonsense mediated mRNA decay in eukaryotes

slide22

Qualitycontrol of translationfor “non-stop” mRNAs

• Non Stop mRNAs can be generated if the poly(A) tail is added before the stop codons (mistakes by the C&P machinery)

Proteasome

• The Poly(A) tail is translated as a Poly-Lysine tract

• Poly-Lysine tract sticks in the ribosome

exit tunnel because of electrostatic

interactions

• Ltn1 ubiquitinligase attaches polyubiquitin

chains to the nascent polypeptide stuck to the

ribosome

• This marks the aberrant protein

for degradation by the proteasome

Bengston & Joazeiro – Nature 2010 - doi: 10.1038/nature09371

premature termination codons ptc are frequently associated with genetic diseases
Premature termination codons (PTC) are frequently associated with genetic diseases
  • >90% of mutations associated with:
  • Duchenne muscular dystrophy
  • Familial adenomatouspolyposis
  • Hereditary desmoid disease
  • Ataxia telangiectasia
  • Hereditary breast and ovarian cancers
  • Polycystic kidney disease
  • >75% of mutations associated with:
  • Emery-Dreifuss muscular dystrophy
  • Fanconi anemia
  • Non-polyposis colorectalcancer

No detection of the truncated proteins

The corresponding mRNA is unstable and degraded by

a mechanism called Nonsense Mediated mRNA Decay(NMD) coupled to translation

slide24

Simplified view of the mechanism of Nonsense Mediated Decay (NMD)

in eukaryotic cells

Translation and Termination proceed normally

STOP

AUG

m7G

(A)n

“Normal” mRNA

Recruitment of RNA degradative

enzymes that prevent accumulation

of PTC-containing mRNAs

Assembly of a complex

that signals NMD

Upf3

Upf2

Upf1

STOP

AUG

STOP

m7G

(A)n

Ribosome Stalled at

Premature Termination

Codon

Aberrant mRNA

containing premature

stop codon (PTC)

mRNA Destruction