title: Histidylation by yeast HisRS of tRNA or tRNA-like structure relies on residues -1 and 73 but is dependent on the RNA context.
abstract: Residue G-1 and discriminator base C73 are the major histidine identity elements in prokaryotes. Here we evaluate the importance of these two nucleotides in yeast histidine aminoacylation identity. Deletion of G-1 in yeast tRNA(His) transcript leads to a drastic loss of histidylation specificity (about 500-fold). Mutation of discriminator base A73, common to all yeast tRNA(His) species, into G73 has a more moderate but still significant effect with a 22-fold decrease in histidylation specificity. Changes at position 36 in the anticodon loop has negligible effect on histidylation. The role of residues -1 and 73 for specific aminoacylation by yeast HisRS was further investigated by studying the histidylation capacities of seven minihelices derived from the Turnip Yellow Mosaic Virus tRNA-like structure. Changes in the nature of nucleotides -1 and 73 modulate this activity but do not suppress it. The optimal mini-substrate for HisRS presents a G.A mismatch at the position equivalent to residues G-1.A73 in yeast tRNA(His), confirms the importance of this structural feature in yeast histidine identity. The fact that the minisubstrates contain a pseudoknot in which position -1 is mimicked by an internal nucleotide from the pseudoknot highlights further the necessity of a stacking interaction of this position over the amino acid accepting branch of the tRNA during the aminoacylation process. Individual transplantation of G-1 or A73 into yeast tRNA(Asp) transcript improves the histidylation efficiency of the engineered tRNA(Asp). However, a tRNA(Asp) transcript presenting simultaneously both residues G-1 and A73 becomes a less good substrate for HisRS, suggesting the importance of the structural context and/or the presence of antideterminants for an optimal expression of these two identity elements.
authors.affiliation: Unité Propre de Recherche 9002 Structures des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Strasbourg, France.
authors.forename: J
authors.initials: J
authors.lastname: Rudinger
authors.forename: C
authors.initials: C
authors.lastname: Florentz
authors.forename: R
authors.initials: R
authors.lastname: Giegé
mesh_terms: D000926:Anticodon / Q000235:genetics; D001483:Base Sequence; D005813:Genes, Synthetic / Q000235:genetics; D006643:Histidine-tRNA Ligase / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation / Q000502:physiology; D012349:RNA, Transfer, Asp / Q000235:genetics; D012354:RNA, Transfer, His / Q000737:chemistry* / Q000235:genetics*; D012367:RNA, Viral / Q000378:metabolism; D017806:Tymovirus / Q000235:genetics; D015003:Yeasts / Q000201:enzymology
chemical_list: D000926:Anticodon; D012349:RNA, Transfer, Asp; D012354:RNA, Transfer, His; D012367:RNA, Viral; D006643:Histidine-tRNA Ligase