A genome-wide perspective on translation of proteins

A genome-wide perspective on translation of proteins

Jan 2012Regulatory GenomicsLecturer: Prof. Yitzhak Pilpel

Selection of codons might affect:AccuracyThroughput

CostsFolding

RNA-structure

Cc d

dC

cC

Fo

d*C

c*C

Free

ene

rgy

l’c

l’d

k’c

k’d

Cc C

d

The energy landscape of kinetic proofreading

F=Fo*

Selection of codons might affect:AccuracyThroughput

CostsFolding

RNA-structure

No correlation between CAI and protein expression among synthetic genes

Prot

ein

abun

danc

e

Correlation does not imply causality!!

r=0.63

Predicted translation efficiency

Mea

sure

d pr

otei

n ab

unda

nce

(Ghaemmaghami et al. Nature 2003)

Evolutionary

Physiological

Tight RNA structure reduce translationPr

otei

n ab

unda

nce

The tightness at the 5’ matters

Natural sequences too show relaxed structure at 5’ (Tuller PNAS 2010)

Stru

ctur

al ti

ghtn

ess

Stru

ctur

al ti

ghtn

ess

Yet, mRNA structure doesn’t predict expression at all

Structural Tightness

Prot

ein/

mRN

A

Bioinformatics vs. synthetic biology

Bioinformatics

Hundreds of thousands of genes

All passed through natural selection

Synthetic biology

Variability is controlled (few confounding factors)

Maybe we had a wrong (i.e. too simple) model for evaluating effect of codons on TE?

Multiple ribosomes may translate the same message simultaneously

A genome-wide method to measure translation efficiency (Ingolia Science 2009)

Translational response to starvation

Putative new ORFs in viruses

How do we validate the new predictions?

What does it mean to “validate” such predictions??

A genome-wide density profile of ribosomes in yeast

Ingolia et al. Nature 2009

A. B.

C. D.

0 50 100 150 2000.27

0.28

0.29

0.3

0.31E. coli

Distance from ATG

Loca

l tAI

0 50 100 150 2000.44

0.45

0.46

0.47

0.48

0.49S. cerevisiae

Distance from ATG

Loca

l tAI

0 50 100 150 2000.4

0.41

0.42

0.43

0.44C. elegans

Distance from ATG

Loca

l tAI

0 50 100 150 2000.35

0.355

0.36

0.365

0.37

0.375

0.38

0.385D. melanogaster

Distance from ATG

Loca

l tAI

Low initial ramp is conserved in evolution

Avai

labi

lity

of tR

NA

Tuller Cell 2010

1 1.05 1.1 1.15 1.2 1.250.4

0.6

0.8

1

1.2

1.4

1/Effective Speed

Rib

osom

e D

ensi

ty

0.6 0.8 1 1.20.5

1

1.5

1/Effective Speed

Rib

osom

e D

ensi

ty

1

2534

5’ -> 3’

Ribosomal density is explained by computed speed

Fluxi,i+1 = Fluxi+1,i+2

Fluxi,i+1 = vi*Ji

1/vi=Ji

At steady-state

Selection of codons might affect:AccuracyThroughput

CostsFolding

RNA-structure

CAA CAG AAA TCG AAT…

Hypothesis: Traffic control by availability of raw material

The anti-Shine–Dalgarno sequence drives translational pausing and codon choice in bacteria

Gene-Wei Li, Eugene Oh & Jonathan S. WeissmanSystem Biology Retreat 2012

Abstract• a genome-wide analysis of pausing in bacteria by

ribosome profiling.• codons decoded by rare tRNAs do not lead to slow translation

under nutrient-rich conditions.• Shine–Dalgarno(SD) like features cause translational pausing. • pausing is due to hybridization between the mRNA and 16S rRNA

of the translating ribosome.• In protein-coding sequences, internal SD sequences are

disfavoured.

• SD-like sequences are a major determinant of translation rates and a global driving force for the coding of bacterial genomes.

Ribosome Profiling

Ribosomes protect from Micrococcal Nuclease

Per transcript

Motivation• ribosome occupancy is highly variable across

coding regions• ribosome density often surpasses by more than

tenfold the mean density• Most pauses are uncharacterized.

Where do the pausing come from???

Pausing due to codons usage? NO!*

Serine codons

Why Serine?serine is the first amino acid to be

catabolized by E. coli when sugar is absent

the increased ribosome occupancy might be due to limited serine supply.

 LB medium

glucose-supplemented MOPS medium

the identity of the A-site codon could not account for the large variability in

ribosome density along messages

Pausing are due to Shine–Dalgarno (SD) like features

Codons resemble features in the SD (AGGAGGU in E. coli)

coincides with spacing for ribosome binding sites.

Pausing are due to SD-like features

Is it Elongating or Initiating Ribosomes?

Experiment:Create a cell with: WT-ribosomes, O-

ribosome & oSD-lacZ.

• On oSD-lacZ:– Pausing on SD-like initiation (by WT ribosomes)– NO Pausing on SD-like elongating ribosomes

SD-like oSD-like

SD-lacZ SD-lacZOther Genes

Pausing are of Elongating Ribosomes

oSD-lacZ oSD-lacZOther Genes

SD-like oSD-like

Internal SD sequences are disfavoured

strong SD-like sequences are generally avoided in the coding region

SD-like features affect codon selection

• GAG, AGG, and GGG are all minor codons

• Selection against two consecutive codons that resemble SD sequences

Why pause ribosomes??

Correspondence of protein structure and ribosome pausing

Conclusions and Discussions1. SD-like features explain pausings, not codons2. SD-like features & 16S elongating ribosome

interacation3. SD-like sequneces are disfavored

to optimiaze translation consider peptide sequence

4. Interactions with ribosomes SD-like codons are disfavoured tRNA expression.

5. conserved pausing can be exploited for functional purposes:

– Frameshifting, folding, transcriptional regulation

Towards more sophisticated translation efficiency models

tRNAs may be recycled

CAA CAG AAA TCG AAT… TCG

Due to recycling the local concentration of a rare tRNA might be high in a near-by codon

Codon Order Influences the Speed of Translation in Yeast Cells

Natural genes have a tendency to look like .I.e. if a rare codon appears at a given position it has an elevated tendency to occur again shortly after along the gene

Cannarozzi et al Cell 2010

Selection of codons might affect:AccuracyThroughput

CostsFolding

RNA-structure

Selection of codons might affect:AccuracyThroughput

CostsFolding

RNA-structure

Glu

GAA (14) GAG (2)?

Slow

Fast

Argos et al. Protein Science 1996

Rare codons at domain-boundaries may support folding

Transient ribosomal attenuation coordinates protein synthesis and co-translational folding

Nature Structural & Molecular Biology 16, 274 - 280 (2009)

Due to co-translation-folding a “synonymous mutation caused a disease – changed a fast codon to a slow one disrupted synchrony of translation and folding