A genome-wide perspective on translation of proteins Jan 2012
Regulatory Genomics Lecturer: Prof. Yitzhak Pilpel
Slide 2
Selection of codons might affect: Accuracy Throughput Costs
Folding RNA-structure
Slide 3
C c d dC cC Fo d*C c*C Free energy lc ld kc kd C cC d The
energy landscape of kinetic proofreading F=Fo*
Slide 4
Selection of codons might affect: Accuracy Throughput Costs
Folding RNA-structure
Slide 5
No correlation between CAI and protein expression among
synthetic genes Protein abundance
Slide 6
Correlation does not imply causality!! r=0.63 Predicted
translation efficiency Measured protein abundance (Ghaemmaghami et
al. Nature 2003) Evolutionary Physiological
Slide 7
Tight RNA structure reduce translation Protein abundance
Slide 8
The tightness at the 5 matters
Slide 9
Natural sequences too show relaxed structure at 5 (Tuller PNAS
2010) Structural tightness Structural tightness
Slide 10
Yet, mRNA structure doesnt predict expression at all Structural
Tightness Protein/mRNA
Slide 11
Bioinformatics vs. synthetic biology Bioinformatics Hundreds of
thousands of genes All passed through natural selection Synthetic
biology Variability is controlled (few confounding factors)
Slide 12
Maybe we had a wrong (i.e. too simple) model for evaluating
effect of codons on TE?
Slide 13
Multiple ribosomes may translate the same message
simultaneously
Slide 14
A genome-wide method to measure translation efficiency (Ingolia
Science 2009)
Slide 15
Translational response to starvation
Slide 16
Putative new ORFs in viruses How do we validate the new
predictions? What does it mean to validate such predictions??
Slide 17
A genome-wide density profile of ribosomes in yeast Ingolia et
al. Nature 2009
Slide 18
Low initial ramp is conserved in evolution Availability of tRNA
Tuller Cell 2010
Slide 19
5 -> 3 Ribosomal density is explained by computed speed Flux
i,i+1 = Flux i+1,i+2 Flux i,i+1 = v i *J i 1/v i =J i At
steady-state
Slide 20
Selection of codons might affect: Accuracy Throughput Costs
Folding RNA-structure
Slide 21
CAACAGAAATCGAAT Hypothesis: Traffic control by availability of
raw material
Slide 22
The anti-ShineDalgarno sequence drives translational pausing
and codon choice in bacteria Gene-Wei Li, Eugene Oh & Jonathan
S. Weissman System Biology Retreat 2012
Slide 23
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. ShineDalgarno(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.
Slide 24
Ribosome Profiling Ribosomes protect from Micrococcal
Nuclease
Slide 25
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???
Slide 26
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
Slide 27
Pausing are due to ShineDalgarno (SD) like features Codons
resemble features in the SD (AGGAGGU in E. coli) coincides with
spacing for ribosome binding sites.
Slide 28
Pausing are due to SD-like features
Slide 29
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
Slide 30
SD-likeoSD-like SD-lacZ Other Genes Pausing are of Elongating
Ribosomes oSD-lacZ Other Genes SD-likeoSD-like
Slide 31
Internal SD sequences are disfavoured strong SD-like sequences
are generally avoided in the coding region
Slide 32
SD-like features affect codon selection GAG, AGG, and GGG are
all minor codons Selection against two consecutive codons that
resemble SD sequences
Slide 33
Why pause ribosomes??
Slide 34
Correspondence of protein structure and ribosome pausing
Slide 35
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
Slide 36
Towards more sophisticated translation efficiency models
Slide 37
tRNAs may be recycled CAACAGAAATCGAAT TCG Due to recycling the
local concentration of a rare tRNA might be high in a near-by
codon
Slide 38
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
Slide 39
Selection of codons might affect: Accuracy Throughput Costs
Folding RNA-structure
Slide 40
Selection of codons might affect: Accuracy Throughput Costs
Folding RNA-structure
Slide 41
Glu GAA (14)GAG (2) ? Slow Fast Argos et al. Protein Science
1996 Rare codons at domain-boundaries may support folding
Slide 42
Transient ribosomal attenuation coordinates protein synthesis
and co-translational folding Nature Structural & Molecular
Biology 16, 274 - 280 (2009)
Slide 43
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
