In this study we have provided, for the first time, a detailed and complete picture of translation in a bacterium. Translatome experiments allowed the determination of the translational status of each mRNA including its ribosome occupancy and ribosome density. In the bacterium under study, L. lactis, the fraction of mRNAs engaged in translation was on average 67% and the mean ribosome density reached 2/5 of the maximal theoretical density. Similar results have been obtained in yeast
[10, 11], although notable differences in the translation process itself (localization, coupling with transcription) and in its regulation (RBS, translation factors) are present between yeast and bacteria. Our data and data obtained in yeast
[10, 11], show that low ribosome density and incomplete mRNAs engaged in translation are two general translational rules in both kingdoms. The low ribosome density indicated for most L. lactis genes that translation is limited at the initiation step. In this particular case, higher ribosome density would result in higher translation level. We have demonstrated that the two translational variables, ribosome occupancy and ribosome density were not constant for all the genes examined here. A high level of variability was indeed demonstrated within the mRNA population, indicating that even if global translation seems to be conserved between microorganisms, this process is also highly transcript-specific. We showed in addition that genes involved in key metabolic pathways exhibited coordinated regulation of ribosome density and ribosome occupancy levels. This result suggests the possibility for L. lactis to use fine translation tuning of selected transcripts during its adaptation to the external environment (e.g. conditions of optimal growth rate). We have also demonstrated that ribosome occupancy was a major determinan of protein level, revealing the high influence of translational regulations in the process coupling gene expression to protein synthesis.
By exploring the diversity of ribosome occupancy and ribosome density values within the mRNA population, systemic key factors involved in translation level regulation were sought. The systemic influence of some factors (codon usage, secondary structure…), previously identified as protein expression regulators of specific mRNAs
[25, 26] or predicted as determinants of translational efficiency by transcriptome-proteome correlation analyses
[2, 27, 28] was seen to act directly on translatome variability. In addition, less expected general factors influencing translation, such as gene function, mRNA half-life and gene length were identified and organized hierarchically. Contribution of these parameters to differentiated translational regulation in natural and stress conditions was also reported in yeast
[10, 11, 29, 30]. Linear covariance models established in L. lactis a link between translational efficiency and gene function. First, genes involved in biosynthesis of amino acids and cofactors exhibited lower ribosome density than others. L. lactis thus adapted its metabolism by attenuating translation of genes involved in these two anabolic pathways. The chemically defined medium used in this study for growing the bacterium contains all the amino acids and vitaminic precursors of the cofactors. In these growth conditions, L. lactis thus adapted its metabolism to ensure cofactor and amino acid supply via their import from external medium rather than via de novo synthesis. This translational attenuation constitutes an adaptive response appropriate to limit energy wastage in the bacterium. We have also observed high ribosome occupancy and high ribosome density for genes of the functional category “regulatory functions”. Important transcriptional regulators were concerned suggesting their efficient translation in vivo. This finding illustrates the capacity of L. lactis cells to coordinate the control of the entire gene expression network through positive translational regulations of key regulators. Mechanisms of transcriptional control by translation have already been reported in E. coli. For mRNAs of some amino acid metabolic enzymes, ribosome deceleration leads to attenuation or antitermination of transcription
[31, 32]. In a more general way, ribosome acceleration and deceleration were shown to directly result in corresponding changes in speed of the RNA polymerase
In addition, an inverse relationship between mRNA stability and both translatome variables was found. As a consequence, less stable mRNAs tended to exhibit rather efficient translation initiation. For initiation-limited unstable genes, higher ribosome density would lead to higher level of translation. mRNA decay and translation would thus act in an antagonistic manner on gene expression regulation in L. lactis. These results are in contrast with what was observed in yeast
. A positive relationship between ribosome density and mRNA half-life was expected in L. lactis, assuming the protective effect of ribosomes against RNases. The absence of such a positive correlation indicates that protection of mRNA against degradation should be more related to ribosome position than to ribosome density. In B. subtilis, mRNA protection by ribosomes located far upstream of cleavage sites (without direct shielding of these sites as required in E. coli) was previously demonstrated
Translation in L. lactis was strongly dependent on coding sequence length, ribosome density being negatively correlated with gene length. Such an inverse correlation between ribosome density and gene length was also reported in yeast
[10, 11]. It would be interesting to verify if an excess of ribosomes at the beginning of the coding sequence as reported in yeast
[35, 36] is also present in L. lactis to explain why short mRNAs tended to present higher ribosome density. However, contrary to yeast
, a positive correlation was obtained between ribosome occupancy and gene length. This correlation is probably necessary in bacteria to counteract the negative ribosome density effect in order to not penalize translation of long important mRNAs. In L. lactis the longest genes represent major functions involved for example in replication, energy metabolism or transport (enrichment results not shown). But more generally, in bacteria, rather long mRNAs are expected in comparison to yeast as a consequence of numerous polycistronic structures.
Using our translatome data, the genome-scale regulation of the initiation and elongation steps was analyzed. mRNA folding was identified as a key determinant of ribosome occupancy. Site-directed experiments at the initiation region have previously demonstrated in E. coli and in L. lactis the association between mRNA folding and translation initiation efficiency
[37, 38]. mRNA folding is expected to interact with ribosome binding site accessibility and modify individual gene expression as demonstrated in E. coli[25, 39]. In addition, for genes with high ribosome occupancy, we have identified a conserved nucleotide pattern (sometimes forming inverted repeat sequences) upstream of the start codon. It is thus tempting to speculate that these sequence features could modulate ribosome binding. Specific translation-enhancing sequences upstream of the start codon have been reported in E. coli and proposed to act as additional interaction sites with either 16S rRNA or S1 ribosomal protein
[40, 41]. However, L. lactis 16S rRNA does not contain a complementary sequence to our motif. In addition, as in other gram-positive bacteria
, no homologous protein of the ribosomal S1 protein was found in L. lactis. Thus, mechanisms of enhancement of translation initiation based on improved mRNA-rRNA base pairing or on increased ribosomal protein-mRNA interaction seem unlikely in L. lactis. Alternatively, the conserved nucleotide pattern found in L. lactis could be involved in secondary structure stability playing a role in translation initiation efficiency
. In yeast, specific base usages around the start codon for genes with high ribosome occupancy were also reported
. Here two determinants of the initiation step were identified; they could be thus involved in the initiation limitation of translation of most of L. lactis genes. Nevertheless, two factors of the elongation step, codon usage and amino acid composition, were shown to influence translation in L. lactis (both at the level of ribosome density). CAI is widely accepted as the key factor in the determination of the elongation rate
[4, 27]. Therefore, our results indicate that for some genes the elongation step could also contribute to translation control corroborating the current point of view of mixed control of translation by initiation and elongation
[4, 19, 27, 28, 36].
To further explore translation regulation, local mRNA ribosome density could be explored via ribosome density mapping or ribosome footprint experiments
[43–45]. Comparison of ribosome density in the 5’UTR of mRNAs exhibiting high or low ribosome occupancies could help to confirm the link between the sequence signatures identified in this work and efficient ribosome binding. The determination of local ribosome densities within a coding sequence could contribute to a better understanding of the relationship between ribosome density and translation efficiency.