The reliability of in Vivo structure-function analysis of tRNA aminoacylation

Modulation of tRNA Ala identity by inorganic pyrophosphatase

“…When aminoacylation reactions by AlaRS are supplemented with PPase to increase the forward rate and EF-Tu⅐GTP to reduce the deacylation rate, high levels of aminoacyl tRNA can be observed for all but the most deleterious tRNa Ala mutations, which agrees with the previous observation that high levels of aa-tRNA are observed in vivo for the same mutations (16). This result in turn explains why Ala is inserted efficiently into proteins by tRNA Ala suppressors containing most identity mutations (17). Thus, tRNAs do not necessarily need to be good substrates in vitro to achieve high levels of aminoacylation in vivo.…”

“…The deleterious effect of mutations of this base pair significantly exceeds those of other tRNA Ala recognition elements (8,9,(12)(13)(14)(15) and clearly was indicative of the predominant role of this base pair and in particular the exocyclic amino group of G3 in recognition. In contrast, in vivo experiments showed that many of same mutations in the G3⅐U70 base pair have only a moderate effect on the levels of aminoacylated tRNA Ala in E. coli and consequently on the ability of the mutant tRNAs to function in protein synthesis (16,17). A careful analysis of the in vivo experiments indicates that the observed discrepancy was not caused by the technical problems associated with the determining of tRNA aminoacylation levels in vivo, and therefore it was proposed that the discrepancy in some way was a consequence of differences in reaction conditions for aminoacylation in vitro and in vivo (17).…”

“…In contrast, in vivo experiments showed that many of same mutations in the G3⅐U70 base pair have only a moderate effect on the levels of aminoacylated tRNA Ala in E. coli and consequently on the ability of the mutant tRNAs to function in protein synthesis (16,17). A careful analysis of the in vivo experiments indicates that the observed discrepancy was not caused by the technical problems associated with the determining of tRNA aminoacylation levels in vivo, and therefore it was proposed that the discrepancy in some way was a consequence of differences in reaction conditions for aminoacylation in vitro and in vivo (17). These experiments lead to the idea that the recognition of tRNA Ala depended more on particular properties of the structure of the tRNA acceptor stem than on base-specific interactions with the G3⅐U70 base pair (11,16).…”

The Development of Novel In Vitro Binding Assays to Further Elucidate the Role of tRNAs in Protein Synthesis

“…In vitro assays showed that mutations within the recognition element drastically reduced aminoacylation (Beuning et al, 1997;Frankyln & Schimmel, 1989;Hou & Schimmel, 1988;McClain et al, 1988;Musier-Forsyth et al, 1991). While in vivo experiments indicated that mutations within the recognition element had a minimal effect (Gabriel et al, 1996;McClain et al, 1999). Despite advancements in in vitro and in vivo assays, a number of discrepancies between the two formats remain.…”

A reduced level of charged tRNAArgmnm5UCU triggers the wild-type peptidyl-tRNA to frameshift

“…Frameshifting induced by mutated arginine tRNA www.rnajournal.org cells under steady-state growth (Varshney et al 1991;Kruger and Sorensen 1998;McClain et al 1999). Visual analysis of the bands in argU2346 (C56U), argU2344 (C61U), argU2343 (G53A), and argU2345 (G39A) mutants clearly indicated that in those samples the amount of uncharged tRNA Arg mnm 5 UCU form increased.…”