4Transfer RNAtRNAs are the small RNAs that link an amino-acid to the peptide sequenceThey have a special palindromic structureThey are amino acid specific AND codon « specific » (wooble)They differ greatly in number in the cell (from ~100 to ~5000 for a given amino acid)
6Differential usage of synonymous codons at the genome scale
7Causes of the codon bias Non-selective causes of the codon biasMutation biases (e. g. towards high/low G+C)Strand bias on the chromosome (GT bias)Selective causes of the codon bias:Translation efficiencyTranslation accuracyCodon-anticodon selection ?Codon robustness ?
8tRNA concentration correlates to codon bias Dong et al. (1996) J. Mol. Biol. 260:649
10Motivations of the project Aim: clustering the genes of an organism according to their codon biasBiological interests:Functional analysis of the groups of genesRole of codon bias in the chromosome structurationComparison of the genome organization between speciesInference of some codon bias causes from the classification
11Previous results Methods: correspondance analysis 2 main sub-groups of genes identified in multiple organisms:Highly expressedHorizontal transfer genesMethodological difficulties:Choice of the number of groupsChoice of the distanceKunst et al. (1997), Nature 390:249
12Key idea about the method: the optimization criteria Each group is defined by the probability distribution of codon usage generated by the genes it containsA good classification is one which maximize the gain of information on these probability distributions, relative to a uniform prior distribution
14Key idea about the method: selection of the number of groups The good number of groups is the one maximizing the average stability of genes attribution inside the groups, relative to the expected stability in absence of structure (random case)
18Gene function is correlated with codon bias Highly expressed genes, translation and ribosomal proteins : COG J (9/22).Unknown genes, pathogenicity islands and horizontally transfered genes : COG - (17/19).Metabolism (synthesis & transport) : COG C (4/6), E (7/4) et F (7).Membrane and carbohydrate metabolism genes : COG G (6) et M (3/3).B. subtilis only -- Motility genes : COG N (5).
19Anabolic genes are grouped on the lagging strand
20Replication and transcription machineries collisions Anabolic genes are usually transcribed when no replication occurs=> being on the lagging strand is not counter-selected.Mirkin & Mirkin (2005) Mol Cell Biol. 25(3): 888
26Motivations of the project Understanding the presence of tRNAs inside bacteriophagesCorrelation to the host or phage codon bias?Differences between lytic and temperate phages?Selection acting on tRNA acquisition and implications for phage evolution?
27Acquisition of tRNA sequences by bacteriophages Lysogenic phages are known to insert in microbial genomes in tRNA sequences=> Imprecise excision could explain the acquisition of tRNA sequencesLytic phages cause liberation of the host genetic material after cell lysis=> Acquisition of tRNAs sequences in the surrounding media or neighbour hosts
28Datas Beginning : 200 DNA phage genomes, 23 hosts, 240 tRNAs Taken out :Non sequenced hostsPhages genomes without tRNAstRNAs inserted in prophagic regionsPhages having tRNAs their host do not haveFinal dataset :37 phages, 15 hosts, 169 tRNAs(6 duplicates, 1 triplet)
30Correlation of host and phages codon bias < R > = 0.77 0.27 real data< R > = 0.38 0.42 phage-random host=> Codon usage is correlated between the host and the phage< R > = 0.83 0.14 real data - Temperate< R > = 0.61 0.39 real data - Lytic=> The correlations are higher in temperate phages
31Phage codon frequency distribution is related to tRNA content
32First conclusionsLytic phages have a codon usage less similar to the one of their hosts when compared to temperate phagesLytic phages have more tRNAs than temperate onesCodon usage is more biased in lytic phages than in temperate onesBoth seem to have tRNAs corresponding to the codons they use more
33Random uptake hypothesis tRNA content of host matches codon biasCodon bias of phage matches the host one’s=> No need for the phage to have tRNAs !Random uptake hypothesis: the tRNA content of a phage should be proportional to its host tRNA content, and so would be indirectly correlated to the codon bias of the phage
34Statistical tests of the random uptake hypothesis Significance for high values of <f>: p = 0.68No specific enrichment in tRNAs for the phage high frequency codonsSignificance for high values of <∆f>: p <Significant enrichment in tRNAs for the codons the phage uses more than its host
35Modelisation of the acquisition and loss processes GainLoss
36Inference of the parameters by maximum likelihood ProbabilityLikelihood ofthe real data,given the modelMaximum likelihoodMost probable
37Evolutive processes tested Selection based on:Frequency of usage of the corresponding codon in the phage genome (+)Frequency of usage of the corresponding codon in the host genome (-)Difference of codon usage frequencies between phage and host genome (+)Duplication of tRNA on the phage genome
38Master model equation results Selection based on the phage frequency of codon usage is non significant (p=0.15)Selection based on the rarity of the codon in the host genome is slightly significant (p=0.018 before Bonferroni correction)Selection based on the difference of frequencies of codon usage between phage and host is highly significant (p<2.10-7)The tRNA duplication hypothesis has to be rejected
39Adaptative selection of tRNAs? Selection relative to the phage codon usage only could lead to a static tRNA content, and could be non-optimal after an host changeSelection relative to the host codon usage only does not take into account the quick phage sequence evolutionSelection needs to take both into account to be adaptative and gives rise to a useful tRNA content
40ConclusionsTranslational selection is a strong pressure acting on phage tRNA contenttRNA content among phages is optimized to compensate for differences between host and phage codon usageThis pressure is more important in lytic phages
41Acknowledgements (II) Massimo VergassolaEduardo RochaThe committee membersYves CharonGuillaume CambrayAymeric Fouquier d’HerouelAll the family and friends who came today!
44Tests of the algorithm (II) High CAI genes share the same codon bias:32/59 in group 1 of B. subtilis33/33 in group 1 of E. coliGenes in the same operon or pathway tend to belong to the same group
45Transcription and translation From Miller et al., 1970, Science 169:392
46Translation regulation and synchronization by tRNA recycling Gene Gene Gene 3
47Recycling phenomenon analysis On average, tRNA recycling should not increase translation speedRecycling could induce a coupling between close ribosomes, allowing for protein synthesis synchronizationSynthetases are the limiting factor as they prevent in most cases a tRNA used by a ribosome to be re-employed by another close one