A genome-wide perspective on
translation of proteins
Jan 2012
Regulatory Genomics
Lecturer: Prof. Yitzhak Pilpel
Selection of codons might affect:
Accuracy
Throughput
RNA-structure
Costs
Folding
The energy landscape of kinetic proofreading
l’c
Free energy
d*C
c*C
l’d
c d
C
k’d
k’c
dC
cC
Fo
c
C
F=Fo*
C
d
Selection of codons might affect:
Accuracy
Throughput
RNA-structure
Costs
Folding
Protein
abundance
No correlation between CAI and protein
expression among synthetic genes
Correlation does not imply causality!!
Measured protein
abundance
r=0.63
Predicted translation efficiency
(Ghaemmaghami et al. Nature 2003)
Protein
abundance
Tight RNA structure reduce translation
The tightness at the 5’ matters
Natural sequences too show relaxed structure at 5’
Structural
tightness
Structural
tightness
(Tuller PNAS 2010)
Protein/mRNA
Yet, mRNA structure doesn’t predict expression at all
Structural Tightness
Bioinformatics vs. synthetic biology
Bioinformatics
Synthetic biology
Hundreds of thousands of
genes
Variability is controlled (few
confounding factors)
All passed through natural
selection
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
Low initial ramp is conserved in evolution
A.
B.
D. melanogaster
of tRNA
Availability
Local tAI
0.385
0.38
Local tAI
0.375
0.37
0.365
0.36
0.355
0.35
0
50
100
150
0.43
0.42
0.41
0.4
0
200
S. cerevisiae
0.31
0.49
0.48
100
150
200
D.
E. coli
0.3
Local tAI
Local tAI
50
Distance from ATG
Distance from ATG
C.
C. elegans
0.44
0.47
0.46
0.29
0.28
0.45
0.44
0
50
100
150
Distance from ATG
Tuller Cell 2010
200
0.27
0
50
100
150
Distance from ATG
200
Ribosomal density is explained by computed speed
1.4
1.2
Fluxi,i+1 = vi*Ji
1
Ribosome Density
Ribosome Density
5’ -> 3’
0.8
0.6
0.4
1
1.05
1.1
1.15
1.5
1
34
Fluxi,i+1 = Fluxi+1,i+2
5
At steady-state
2
1
0.5
0.6 0.8
1
1.2
1/Effective Speed
1.2
1/Effective Speed
1.25
1/vi=Ji
Selection of codons might affect:
Accuracy
Throughput
RNA-structure
Costs
Folding
Hypothesis: Traffic control by availability of raw
material
…
CAA CAG AAA TCG AAT
The anti-Shine–Dalgarno sequence
drives translational pausing and codon
choice in bacteria
System Biology Retreat 2012
Gene-Wei Li, Eugene Oh & Jonathan S. Weissman
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
Per transcript
𝑃 𝑓𝑜𝑜𝑡𝑝𝑟𝑖𝑛𝑡 ∝ 𝑡𝑖𝑚𝑒
⇒ 𝐻𝑖𝑔ℎ𝑒𝑟 𝑅𝑖𝑏𝑜𝑠𝑜𝑚𝑒 𝑜𝑐𝑐𝑢𝑝𝑎𝑛𝑐𝑦
→ 𝑙𝑜𝑛𝑔𝑒𝑟 𝑑𝑤𝑒𝑙𝑙 𝑡𝑖𝑚𝑒
 Ribosomes protect from
Micrococcal Nuclease
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!*
LB medium
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.
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
Pausing are of Elongating Ribosomes
SD-lacZ
SD-like
oSD-like
oSD-lacZ
SD-like
oSD-like
SD-lacZ
Other Genes
oSD-lacZ
Other Genes
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 Discussions
1. SD-like features explain pausings, not codons
2. SD-like features & 16S elongating ribosome interacation
3. 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:
Accuracy
Throughput
RNA-structure
Costs
Folding
Selection of codons might affect:
Accuracy
Throughput
RNA-structure
Costs
Folding
Rare codons at domain-boundaries may support folding
Glu
?
GAA (14)
Slow
Fast
GAG (2)
Argos et al. Protein Science 1996
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