Proline codon pair selection determines ribosome pausing strength and translation efficiency in bacteria

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.

Decoding of translation‐regulating entities reveals heterogeneous translation deficiency patterns in cellular senescence

“…Stalling on proline codons was observed on all three ribosome sites, while the previous or next codon again encoded proline (Figure 3b). This is in line with the recent observation that diproline (Pro-Pro) motifs may be responsible for paused ribosomes (Krafczyk et al, 2021). Overall differences in ribosome stalling between H 2 O 2 -treated HeLa cells and controls were found to be insignificant (p > 0.05; Figure 3c, left).…”

The Bacterial ClpXP-ClpB Family Is Enriched with RNA-Binding Protein Complexes

“…In CLPP-null mouse heart complexomics, the disperse co-migration of accumulated CLPX with its accumulated interactions partners such as GFM1, GFM2, POLDIP2, GRSF1, LRPPRC, and FASTKD4 is best explained by their association with partially insoluble, misfolded, incomplete translation products of varying sizes with trapped misprocessed mRNA/tRNA, which cannot be degraded intra-mitochondrially in an efficient manner due to the absence of CLPP. It has been shown for bacterial translation that the peptide bond formation is especially slow with proline and that two adjacent prolines can even cause ribosome stalling [ 127 , 128 ]. Therefore, it is interesting to note that human/mouse sequences of COX1 as a key element of the rate-limiting respiratory complex-IV contain 2 double-prolines and 1 triple-proline (UniProt U5YWV7/Q9MD68), perhaps explaining why complex-IV biogenesis facilitators such as TACO1-SURF1-COX15 are prominent among CLPB and CLPXP interactors and the pathway of Cytochrome-C oxidase deficiency disease is enriched among CLPP interactions (see Figure 1 , Figure 2 and Figure 3 ).…”

Omics profiling identifies the regulatory functions of the MAPK/ERK pathway in nephron progenitor metabolism

“…To identify the wider spectrum of metabolic changes, an untargeted approach was applied to control and MEK-inhibited mK4 cells. Comparison of the untargeted tandem mass spectrometry (MS2) spectrum patterns with an online MS database revealed a significant decrease of 34 metabolites, including polyamines (N-acetylputrescine), amino acids such as glutamine and its metabolites, L-aspartic acid, leucine, proline, threonine and tryptophan ( Table S2 ) ( Melnikov et al, 2016 ; Krafczyk et al, 2021 ). Also, methionine, which was recently suggested to contribute to NP proliferation ( Makayes et al, 2021 ), was diminished upon MAPK/ERK deficiency.…”