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Translation is a central cellular process and is optimized for speed and fidelity. The speed of translation of a single codon depends on the concentration of aminoacyl-tRNAs. Here, we used microarray-based approaches to analyze the charging levels of tRNAs in Escherichia coli growing at different growth rates. Strikingly, we observed a non-uniform aminoacylation of tRNAs in complex media. In contrast, in minimal medium, the level of aminoacyl-tRNAs is more uniform and rises to approximately 60%. Particularly, the charging level of tRNA(Ser), tRNA(Cys), tRNA(Thr) and tRNA(His) is below 50% in complex medium and their aminoacylation levels mirror the degree that amino acids inhibit growth when individually added to minimal medium. Serine is among the most toxic amino acids for bacteria and tRNAs(Ser) exhibit the lowest charging levels, below 10%, at high growth rate although intracellular serine concentration is plentiful. As a result some serine codons are among the most slowly translated codons. A large fraction of the serine is most likely degraded by L-serine-deaminase, which competes with the seryl-tRNA-synthetase that charges the tRNAs(Ser). These results indicate that the level of aminoacylation in complex media might be a competition between charging for translation and degradation of amino acids that inhibit growth.
Transfer RNAs (tRNAs) through their abundance and modification pattern significantly influence protein translation. Here, we present a systematic analysis of the tRNAome of Lactococcus lactis. Using the next-generation sequencing approach, we identified 40 tRNAs which carry 16 different post-transcriptional modifications as revealed by mass spectrometry analysis. While small modifications are located in the tRNA body, hypermodified nucleotides are mainly present in the anticodon loop, which through wobbling expand the decoding potential of the tRNAs. Using tRNA-based microarrays, we also determined the dynamics in tRNA abundance upon changes in the growth rate and heterologous protein overexpression stress. With a fourfold increase in the growth rate, the relative abundance of tRNAs cognate to low abundance codons decrease, while the tRNAs cognate to major codons remain mostly unchanged. Significant changes in the tRNA abundances are observed upon protein overexpression stress, which does not correlate with the codon usage of the overexpressed gene but rather reflects the altered expression of housekeeping genes.