title: Structural and functional accommodation of nucleotide variations at a conserved tRNA tertiary base pair.
abstract: The U8:A14 tertiary base pair of transfer RNAs (tRNAs) stabilizes the sharp turn from the acceptor stem to the dihydrouridine stem. This tertiary base pair is important for the overall L-shaped tRNA structure. Inspection of tRNA sequences shows that U8:A14 is highly conserved. However, variations of U8:A14 are found in natural sequences. This raises the question of whether all 16 permutations of U8:A14 can be accommodated by a single tRNA sequence framework and by the bacterial translational apparatus. Here we expressed the wild type and 15 variants of U8:A14 of an alanine tRNA amber suppressor in Escherichia coli and tested the ability of each to suppress an amber mutation. We showed that 12 of the 15 variants are functional suppressors (sup+) and 3 are nonfunctional (sup-). Of the 12 functional suppressors, the G8:G14 variant is the most efficient suppressor, whose suppression efficiency is indistinguishable from that of the wild type. Analysis of tRNA structure with chemical probes and the lead-cleavage reaction, however, showed a distinct difference between the G8:G14 variant and the wild type. Thus, two different structures of E. coli tRNAAla/CUA share an identical functional phenotype in protein synthesis. The remaining 11 sup+ variants with reduced suppression efficiencies are likely to have other structural variations. We suggest that the variations of these sup+ mutants are structurally and functionally accommodated by the bacterial translational apparatus. In contrast, the three sup- mutants harbor variations that alter the backbone structure in the corner of the L. These variations are likely to reduce the stability of the tRNA inside the cell or, among others, to interfere with the ability of the tRNA to functionally interact with elongation factor Tu and with the ribosome.
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis; D001482:Base Composition; D001483:Base Sequence; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012344:RNA, Transfer, Ala / Q000737:chemistry* / Q000235:genetics*; D012265:Ribonucleotides; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D012344:RNA, Transfer, Ala; D012265:Ribonucleotides; D001616:beta-Galactosidase