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Abstract
The speed of mRNA translation depends in part on the amino acid to be incorporated into the nascent chain. Peptide bond formation is especially slow with proline and two adjacent prolines can even cause ribosome stalling. While previous studies focused on how the amino acid context of a Pro-Pro motif determines the stalling strength, we extend this question to the mRNA level. Bioinformatics analysis of the Escherichia coli genome revealed significantly differing codon usage between single and consecutive prolines. We therefore developed a luminescence reporter to detect ribosome pausing in living cells, enabling us to dissect the roles of codon choice and tRNA selection as well as to explain the genome scale observations. Specifically, we found a strong selective pressure against CCC/U-C, a sequon causing ribosomal frameshifting even under wild-type conditions. On the other hand, translation efficiency as positive evolutionary driving force led to an overrepresentation of CCG. This codon is not only translated the fastest, but the corresponding prolyl-tRNA reaches almost saturating levels. By contrast, CCA, for which the cognate prolyl-tRNA amounts are limiting, is used to regulate pausing strength. Thus, codon selection both in discrete positions but especially in proline codon pairs can tune protein copy numbers.
Highlights
The speed of mRNA translation depends in part on the amino acid to be incorporated into the nascent chain
Our study started with a bioinformatics analysis, in which we investigated whether codon usage differs between single prolines and proline pairs in the proteome of E. coli MG1655 (Figs. 2 and 3)
Selection against slowly translating proline codon pairs is not restricted to E. coli: Out of 15 bacterial genomes with a broad range of GC-content values CCC and CCU are disfavored in 13 and 11 genomes, respectively (Fig. S1 and Supplementary data file S1)
Summary
The speed of mRNA translation depends in part on the amino acid to be incorporated into the nascent chain. CCA, for which the cognate prolyl-tRNA amounts are limiting, is used to regulate pausing strength Codon selection both in discrete positions but especially in proline codon pairs can tune protein copy numbers. Is peptide bond formation with proline the slowest compared to all other proteinogenic amino acid[3,4,5], but ribosomes can even be arrested when translating stretches of proline residues[6,7,8]. In Escherichia coli every third protein contains at least one polyproline motif (PP-motif, at least diproline)[11] and in Streptomyces species there is more than one PP-motif per protein on average[12] The explanation for this apparent oddity is the existence of a ubiquitous elongation factor (termed EF-P in bacteria and a/eIF5A in archaea/eukaryotes) that alleviates ribosome stalling[13,14,15,16]. We have comprehensively investigated how the interplay of codon choice and tRNA abundance affect the translation of PP-motifs
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