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Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis

Borg, Anneli, Ehrenberg, Måns
Journal of molecular biology 2015 v.427 no.9 pp. 1835-1847
Gibbs free energy, bacterial proteins, chemical bonding, magnesium, messenger RNA, open reading frames, peptide elongation factors, protein synthesis, ribosomal RNA, ribosomes, start codon, transfer RNA
Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30s⁻¹ at 1mM free Mg²⁺ concentration to the detrimentally low value of 1s⁻¹ at 6mM free Mg²⁺ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg²⁺ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0–6kcal/mol. Based on these experiments, we discuss the optimal choice of Mg²⁺ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account.