Structure and function of unmodified E. coli valine-tRNA
The effects of nucleoside modifications on E. coli tRNA[superscript]Val structure have been probed by imino [superscript]1H NMR. The NMR data shows that the structure of in vitro transcribed (unmodified) and native (modified) tRNA[superscript] Val are very similar in 15 mM Mg[superscript]2+. Temperature dependence of the spectra reveals that nucleoside modifications stabilize tertiary interactions between T and D loops. On removal of Mg[superscript]2+, unmodified tRNA[superscript] Val undergoes remarkable structural changes which are not observed in native tRNA[superscript] Val. There is near total disruption of the D stem and of tertiary interactions. 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 decreased strength of Mg[superscript]2+ binding in unmodified tRNA[superscript] Val. However, in the absence of Mg[superscript]2+, mutant G38 has a stable structural core in the acceptor stem and the anticodon stem, unlike that found in wild type tRNA[superscript] Val. 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. tRNA[superscript] Val position 35 mutants do not inhibit aminoacylation of wild type tRNA[superscript] Val suggesting that valyl-tRNA synthetase discrimination is affinity-based;[superscript]19F NMR studies of FU-tRNA[superscript] Val mutants of positions 32 and 38 provide evidences for GU base pair formation between G38 and U32, but not between G32 and U38. Unexplained chemical shift positions for residues FU32 and FU33 are found in the [superscript]19F NMR spectrum of mutant U32C38;Most resonances in the [superscript]1H NMR spectrum of codon containing tetranucleotide GUAA have been assigned. Transfer NOE experiments have been initiated to determine the structure of GUAA when bound to tRNA[superscript] Val.