Structure and function of unmodified E. coli valine-tRNA

Abstract

The clFects of nucleoside modifications on E. coii tllNA^^' structure have be(Mi probed by imino Hi NMR. The NMR data shows that the structure of in oilro transcribed (unmodified) and native (modified) tRNA^^' are very similar in 15 iiiM Mg-^. Temperature dependence of the spectra reveals that nucleoside modifications stabilize tertiary interactions between T and D loops. On removal of Mg^', unmod­ ified tRNA^'^' undergoes remarkable structural changes which are not observed in native tRNA^^''^'. There is near total disruption of the D stem and of tertiary in­ teractions. A new strong interaction occurs between the A6U67 base pair (acceptor stem) and the G50U64 wobble base pair (T .stem). This interaction remains intact and forms a stable structural core even at 60 °C. These conformational changes are correlated with the decrea.sed strength of Mg^^ binding in unmodified tRNA^'^'. However, in the absence of Mg*^, mutant G38 has a stable structural core in the acceptor stem and the anticodon stem, unlike that found in wild type tRNA^^'. Such structural changes are caused by a base pair formation between G38 and C32. This may also account for the reduction in catalytic efficiency of G38 mutant. tRNyX^'*' position .35 mutants do not inhibit aminoacylation of wild type tRNA^'^', suggesting that valyl-tRNA synthetase di.scriniination is affinity-based. NMR studies of FU-tRNA^^' mutants of positions 32 and 38 provide evi­