For example, 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. However, consecutive prolines occur frequently in eukaryotic and prokaryotic proteomes 9, 10. Not only 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. However, all these unique features come at a price. Peptide stretches enriched in prolines can even form a distinct type of secondary structure, the so called polyproline helix 2. Its pyrrolidine ring makes proline conformationally rigid and thus it can shape protein structure: depending on its configuration- cis or trans-the binding axis rotation of amide bonds changes with major consequences for folding 1. It is the only n-alkyl amino acid and thus has unique chemical properties. Proline has a set of characteristics that is not found in other proteinogenic amino acids. Thus, codon selection both in discrete positions but especially in proline codon pairs can tune protein copy numbers. By contrast, CCA, for which the cognate prolyl-tRNA amounts are limiting, is used to regulate pausing strength. This codon is not only translated the fastest, but the corresponding prolyl-tRNA reaches almost saturating levels. On the other hand, translation efficiency as positive evolutionary driving force led to an overrepresentation of CCG. Specifically, we found a strong selective pressure against CCC/U-C, a sequon causing ribosomal frameshifting even under wild-type conditions. 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. Bioinformatics analysis of the Escherichia coli genome revealed significantly differing codon usage between single and consecutive prolines. 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. Peptide bond formation is especially slow with proline and two adjacent prolines can even cause ribosome stalling. The speed of mRNA translation depends in part on the amino acid to be incorporated into the nascent chain.
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