title: Distinctive acceptor-end structure and other determinants of Escherichia coli tRNAPro identity.
abstract: The previously uncharacterized determinants of the specificity of tRNAPro for aminoacylation (tRNAPro identity) were defined by a computer comparison of all Escherichia coli tRNA sequences and tested by a functional analysis of amber suppressor tRNAs in vivo. We determined the amino acid specificity of tRNA by sequencing a suppressed protein and the aminoacylation efficiency of tRNA by examining the steady-state level of aminoacyl-tRNA. On substituting nucleotides derived from the acceptor end and variable pocket of tRNAPro for the corresponding nucleotides in a tRNAPhe gene, the identity of the resulting tRNA changed substantially but incompletely to that of tRNAPro. The redesigned tRNAPhe was weakly active and aminoacyl-tRNA was not detected. Ethyl methanesulfonate mutagenesis of the redesigned tRNAPhe gene produced a mutant with a wobble pair in place of a base pair in the end of the acceptor-stem helix of the transcribed tRNA. This mutant exhibited both a tRNAPro identity and substantial aminoacyl-tRNA. The results speak for the importance of a distinctive conformation in the acceptor-stem helix of tRNAPro for aminoacylation by the prolyl-tRNA synthetase. The anticodon also contributes to tRNAPro identity but is not necessary in vivo.
authors.affiliation: Department of Bacteriology, University of Wisconsin, Madison 53706-1567.
authors.forename: W H
authors.initials: WH
authors.lastname: McClain
authors.forename: J
authors.initials: J
authors.lastname: Schneider
authors.forename: K
authors.initials: K
authors.lastname: Gabriel
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism*; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012361:RNA, Transfer, Pro / Q000737:chemistry*; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D046249:Transfer RNA Aminoacylation
chemical_list: D012361:RNA, Transfer, Pro; D000604:Amino Acyl-tRNA Synthetases