title: Covariation of a specificity-determining structural motif in an aminoacyl-tRNA synthetase and a tRNA identity element.
abstract: Transfer RNA (tRNA) identity determinants help preserve the specificity of aminoacylation in vivo, and prevent cross-species interactions. Here, we investigate covariation between the discriminator base (N73) element in histidine tRNAs and residues in the histidyl-tRNA synthetase (HisRS) motif 2 loop. A model of the Escherichia coli HisRS--tRNA(His) complex predicts an interaction between the prokaryotic conserved glutamine 118 of the motif 2 loop and cytosine 73. The substitution of Gln 118 in motif 2 with glutamate decreased discrimination between cytosine and uracil some 50-fold, but left overall rates of adenylation and aminoacylation unaffected. By contrast, substitutions at neighboring Glu 115 and Arg 121 affected both adenylation and aminoacylation, consistent with their predicted involvement in both half-reactions. Additional evidence for the involvement of the motif 2 loop was provided by functional analysis of a hybrid Saccharomyces cerevisiae-- E. coli HisRS possessing the 11 amino acid motif 2 loop of the yeast enzyme. Despite an overall decreased activity of nearly 1000-fold relative to the E. coli enzyme, the chimera nevertheless exhibited a modest preference for the yeast tRNA(His) over the E. coli tRNA, and preferred wild-type yeast tRNA(His) to a variant with C at the discriminator position. These experiments suggest that part of, but not all of, the specificity is provided by the motif 2 loop. The close interaction between enzyme loop and RNA sequence elements suggested by these experiments reflects a covariation between enzyme and tRNA that may have acted to preserve aminoacylation fidelity over evolutionary time.
authors.affiliation: Department of Biochemistry, University of Vermont, Health Sciences Complex, Burlington, Vermont 05405, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Hawko
authors.forename: C S
authors.initials: CS
authors.lastname: Francklyn
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001226:Aspartate-tRNA Ligase / Q000737:chemistry / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006643:Histidine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012349:RNA, Transfer, Asp / Q000737:chemistry / Q000378:metabolism; D012354:RNA, Transfer, His / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013045:Species Specificity; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D012349:RNA, Transfer, Asp; D012354:RNA, Transfer, His; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D001226:Aspartate-tRNA Ligase; D006643:Histidine-tRNA Ligase