Enhanced proofreading attenuates initial selection error hot spots in genetic code translation by transfer RNAs
(English)Manuscript (preprint) (Other academic)
A system for cell free protein synthesis with E. coli components of high purity was used in conjunction with fast kinetics quench-flow measurements to characterize the accuracy of peptie bond formation by ribosomes with initiator tRNAfMet in P site and different codons in the A site. We used Glu-tRNAGlu, Lys-tRNALys and Phe-tRNAPhe in ternary complexes with EF-Tu and GTP to select ribosomes programmed with their respective cognate codons in competition with ribosomes containing near-cognate codons. Variation of the free Mg2+ concentration in the in vitro buffer system was used to calibrate its accuracy to that of codon selection by Glu-tRNAGlu in living E. coli cells, previously estimated from the residual activity of a beta-galactosidase mutant in which the codon for an essential Glu had been altered to near cognate codons. At 2.3 mM free Mg2+ concentration, the accuracy in the living cell agreed with that in the test tube, a feature making our biochemistry directly relevant to bacterial physiology. We found that the total accuracy of tRNA selection varied by five orders of magnitude depending on the type of tRNA, type of mismatch and mismatched codon position. We partitioned the total accuracy into initial selection of ternary complex before GTP hydrolysis on EF-Tu and proofreading selection of aminoacyl-tRNA after GTP hydrolysis. We found the contribution of proofreading to be strongly positively correlated with the accuracy of initial selection in its high range. As initial selection decreased further the proofreading contribution to accuracy increased, rather than decreased, a feature neutralizing potentially disastrous missense error hot spots associated with, in particular, tRNAGlu.
protein synthesis, genetic code, misreading, error hot spots, kinetics, initial selection, proofreading, total accuracy
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-264957OAI: oai:DiVA.org:uu-264957DiVA: diva2:862232