A genome-wide perspective on translation of proteins Jan 2012 Regulatory Genomics Lecturer: Prof. Yitzhak Pilpel

Selection of codons might affect: Accuracy Throughput Costs Folding RNA-structure

C c d dC cC Fo d*C c*C Free energy l’c l’d k’c k’d C cC d The energy landscape of kinetic proofreading F=Fo*

Selection of codons might affect: Accuracy Throughput Costs Folding RNA-structure

No correlation between CAI and protein expression among synthetic genes Protein abundance

Correlation does not imply causality!! r=0.63 Predicted translation efficiency Measured protein abundance (Ghaemmaghami et al. Nature 2003) Evolutionary Physiological

Tight RNA structure reduce translation Protein abundance

The tightness at the 5’ matters

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

Yet, mRNA structure doesn’t predict expression at all Structural Tightness Protein/mRNA

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

Low initial ramp is conserved in evolution Availability of tRNA Tuller Cell 2010

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

Selection of codons might affect: Accuracy Throughput Costs Folding RNA-structure

CAACAGAAATCGAAT … 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. Weissman System 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

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-likeoSD-like SD-lacZ Other Genes Pausing are of Elongating Ribosomes oSD-lacZ Other Genes SD-likeoSD-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 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 CAACAGAAATCGAAT … 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 Costs Folding RNA-structure

Selection of codons might affect: Accuracy Throughput Costs Folding 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, (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