Kinetic Determinants of High-Fidelity tRNA Discrimination on the Ribosome

Abstract

The ribosome selects aminoacyl-tRNA (aa-tRNA) matching to the mRNA codon from the bulk of non-matching aa-tRNAs in two consecutive selection steps, initial selection and proofreading. Here we report the kinetic analysis of selection taking place under conditions where the overall selectivity was close to values observed in vivo and initial selection and proofreading contributed about equally. Comparison of the rate constants shows that the 350-fold difference in stabilities of cognate and near-cognate codon-anticodon complexes is not used for tRNA selection due to high rate of GTP hydrolysis in the cognate complex. tRNA selection at the initial selection step is entirely kinetically controlled and is due to much faster (650-fold) GTP hydrolysis of cognate compared to near-cognate substrate.

Ribosome kinetics and aa-tRNA competition determine rate and fidelity of peptide synthesis

“…Table 4 (Varenne et al, 1984, p. 570) summarizes average synthesis rates for various proteins in E. coli. The rates which have been measured at 24.5 °C (Rate B in their Table 4) compare very closely with our theoretical results, which use the kinetic rates of Gromadski and Rodnina (2004) measured at 20 °C. Heyd and Drew (2003), who also based their model on the mechanism proposed by Rodnina and co-workers, obtained an average translation rate of 2.24 amino acids/s.…”

“…(3) Interference from other ribosomes on the same mRNA is not considered. (4) The translation rates are derived for the kinetic rates as reported by Gromadski and Rodnina (2004). (5) Kinetic rate constants which have not been measured are assigned large values in order not to become rate determining.…”

The pathway to GTPase activation of elongation factor SelB on the ribosome

“…When necessary, quench-flow experiments were performed in a KinTek apparatus. Reactions were quenched with formic acid (25% v/v) and the extent of GTP cleavage was determined by thin layer chromatography and phosphor imaging 57 . The rate of GTP hydrolysis was determined by exponential fitting of the time courses using GraphPad Prism software (GraphPad Software, Inc.); time courses were normalized to the respective reaction end levels.…”

Charting the dynamics of translation

“…The model is agent-based representing explicitly every single mRNA and ribosome. The latter bind to individual transcripts following first order kinetics and then perform a directed random walk with transition rates calculated following the Gromadski-Rodnina model (Gromadski and Rodnina, 2004;Fluitt et al, 2007). Upon termination ribosomes may re-initiate at the same transcript or unbind into the cell volume to rebind to a randomly chosen transcript at a later time again.…”

“…The current best understanding of the factors determining the decoding time goes back to a model by Gromadski and Rodnina (2004). The central element of the model is that cognate aa-tRNA species compete with near matches (the so-called near-cognate aa-tRNA) for access to the ribosome.…”