tRNA structure and ribosomal function. I. tRNA nucleotide 27-43 mutations enhance first position wobble.
Transfer RNA su7 G36 is a derivative of tRNA(Trp) with a 3'GUC anticodon complementary to the glutamine codon CAG. This tRNA requires a normally forbidden G-U wobble at the first codon position to suppress a UAG (amber) termination codon. Measurement of amber suppression by mutated su7 G36 tRNAs and correction for tRNA levels and aminoacylation allowed calculation of KUAG, a linearized index of in vivo ribosomal function. Following saturating mutagenesis of the anticodon arm of su7 G36, screening for UAG suppression using a lacZ reporter yielded tRNAs with up to 40-fold increased first position G-U wobble, judged from KUAG. The parental anticodon helix has minimized this type of miscoding, and virtually all changes in the top base-pair of the anticodon helix, nucleotides (nt) 27-43, increased the error. Thus, misincorporation of amino acids due to aberrant first position wobble is apparently prevented by normal tRNA structure, which is specifically altered by substitution at nt 27-43, the top base-pair of the anticodon helix. All 16 permutations of nt 27-43, the hotspot for increased wobble, were subsequently constructed and compared. Comparison of values for tRNA coding function, tRNA level, and aminoacylation for the 16 suggest that a tRNA conformational change, specifically involving both nt 27-43, differentially affects all these tRNA functions. This conformational alteration, which presumably occurs normally on the ribosome, appears more complex than simple breakage of the normal 27-43 base-pair. We suggest that the change is in the angle and/or flexibility of the tRNA L-shape. Among these 16 tRNAs, efficient wobble is strongly and inversely correlated with good aminoacylation and high tRNA levels; this quality may have been selected. Constraints on the sequences of natural tRNAs suggest that nt 27-43 have effects on function in many tRNAs.
Schultz
D W
DW
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309-0347.
Yarus
M
M
D000926:Anticodon; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005973:Glutamine; D008961:Models, Structural; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics; D012364:RNA, Transfer, Trp / Q000737:chemistry* / Q000235:genetics; D012044:Regression Analysis
D000926:Anticodon; D003062:Codon; D012364:RNA, Transfer, Trp; D005973:Glutamine; D012343:RNA, Transfer