PMID:10387013

Citation	Horowitz, J, Chu, WC, Derrick, WB, Liu, JC, Liu, M and Yue, D (1999) Synthetase recognition determinants of E. coli valine transfer RNA. Biochemistry 38:7737-46
Abstract	We have studied the interactions between Escherichia coli tRNAVal and valyl-tRNA synthetase (ValRS) by enzymatic footprinting with nuclease S1 and ribonuclease V1, and by analysis of the aminoacylation kinetics of mutant tRNAVal transcripts. Valyl-tRNA synthetase specifically protects the anticodon loop, the 3' side of the stacked T-stem/acceptor-stem helix, and the 5' side of the anticodon stem of tRNAVal against cleavage by double- and single-strand-specific nucleases. Increased nuclease susceptibility at the ends of the anticodon- and T-stems in the tRNAVal.ValRS complex is indicative of enzyme-induced conformational changes in the tRNA. The most important synthetase recognition determinants are the middle and 3' anticodon nucleotides (A35 and C36, respectively); G20, in the variable pocket, and G45, in the tRNA central core, are minor recognition elements. The discriminator base, position 73, and the anticodon stem also are recognized by ValRS. Replacing wild-type A73 with G73 reduces the aminoacylation efficiency more than 40-fold. However, the C73 and U73 mutants remain good substrates for ValRS, suggesting that guanosine at position 73 acts as a negative determinant. The amino acid acceptor arm of tRNAVal contains no other synthetase recognition nucleotides, but regular A-type RNA helix geometry in the acceptor stem is essential [Liu, M., et al. (1997) Nucleic Acids Res. 25, 4883-4890]. In the anticodon stem, converting the U29:A41 base pair to C29:G41 reduces the aminoacylation efficiency 50-fold. This is apparently due to the rigidity of the anticodon stem caused by the presence of five consecutive C:G base pairs, since the A29:U41 mutant is readily aminoacylated. Identity switch experiments provide additional evidence for a role of the anticodon stem in synthetase recognition. The valine recognition determinants, A35, C36, A73, G20, G45, and a regular A-RNA acceptor helix are insufficient to transform E. coli tRNAPhe into an effective valine acceptor. Replacing the anticodon stem of tRNAPhe with that of tRNAVal, however, converts the tRNA into a good substrate for ValRS. These experiments confirm G45 as a minor ValRS recognition element.
Links	PubMed Online version:10.1021/bi990490b
Keywords	Anticodon/chemistry; Anticodon/metabolism; Base Sequence; Binding Sites; Endoribonucleases/chemistry; Escherichia coli/enzymology; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; RNA, Transfer, Phe/chemistry; RNA, Transfer, Phe/genetics; RNA, Transfer, Phe/metabolism; RNA, Transfer, Val/chemistry; RNA, Transfer, Val/metabolism; Single-Strand Specific DNA and RNA Endonucleases/chemistry; Valine-tRNA Ligase/chemistry; Valine-tRNA Ligase/genetics; Valine-tRNA Ligase/metabolism
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