Transfer RNA structure and coding specificity. II. A D-arm tertiary interaction that restricts coding range.
We investigated the structural basis of the kinetic effect on coding specificity by the D-arm mutant (G24 to A) of Escherichia coli tRNATrp. A set of tRNA genes with structural alterations in the D-arm was constructed by site-directed mutagenesis in vitro, and we determined the in vivo translational activities of these tRNAs. Our results suggest that a hydrogen-bond donor in the major groove of the D-helix at position 24 is required for the expansion of tRNA wobble coding specificity. From inspection of tRNA crystal structure, we identified a potential new tertiary pairing of base 24 with the base at position 9 (this base links the acceptor and D-stems). We constructed tRNAs with mutations at position 9 and showed that the phenotypes of position 11-24 D-arm mutants are indeed dependent on the identity of base 9. Our analysis of the effects of these mutations on the interactions of tRNA with the ribosome and with aminoacyl-tRNA synthetase suggests that the conformation or conformational dynamics of the middle of the tRNA molecule alters the kinetics of the interaction with the ribosomal coding site. The 9-23 and putative 9-24 tertiaries, and perhaps other normal tertiary interactions in this region, modulate these kinetics to increase or decrease coding specificity.
Smith
D
D
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309.
Yarus
M
M
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D003062:Codon / Q000235:genetics*; D004926:Escherichia coli; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*; D012270:Ribosomes; D013489:Suppression, Genetic
D003062:Codon; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases