Interactions of Escherichia coli SO-187 tRNAIVal with Bacillus stearothermophilus valine-tRNA synthetase studied by13C-NMR

Schweizer, Martin; Olsen, Jay I.; Stolk, Jacob A.; Lee, Y.-C.; Reeves, Patrick M.; Perry, Colin; N, De

doi:10.1016/0167-4781(89)90019-5

Cited by 2 publications

(1 citation statement)

References 16 publications

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“…Loss of intensity of the signal from FU34 is the major effect of synthetase binding on the 19 F spectrum of (FUra)tRNA Val (21). Schweizer and coworkers reported comparable results in 13 C NMR experiments probing the interaction of [4,[5][6][7][8][9][10][11][12][13] C]uracil-labeled E. coli tRNA Val and B. stearothermophilus valyl-tRNA synthetase (41,42). 19 F NMR experiments also provide evidence for synthetase contacts at FU7 and FU67 (21), consistent with the nuclease footprinting results, which show protection by ValRS against nuclease V1 cleavage at the nearby residues 8 and 9 and at 66 (Figures 1 and 2).…”

Section: Resultsmentioning

confidence: 96%

Synthetase Recognition Determinants of E. coli Valine Transfer RNA

et al. 1999

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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.

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Section: Resultsmentioning

confidence: 96%

Synthetase Recognition Determinants of E. coli Valine Transfer RNA

et al. 1999

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Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study

Chu¹,

Horowitz²

1991

Biochemistry

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Interactions of 5-fluorouracil-substituted Escherichia coli tRNAVal with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene [Heck, J.D., & Hatfield, G.W. (1988) J. Biol. Chem. 263, 868-877]. Apparent KM and Vmax values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNAVal. Binding of VRS to (FUra)tRNAVal induces structural perturbations that are reflected in selective changes in the 19F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNAVal along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNAVal, suggesting conformational changes in this part of the molecule. No 19F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNAVal that has been proposed as a common intermediate in the aminoacylation reaction.

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Interactions of Escherichia coli SO-187 tRNAIVal with Bacillus stearothermophilus valine-tRNA synthetase studied by13C-NMR

Cited by 2 publications

References 16 publications

Synthetase Recognition Determinants of E. coli Valine Transfer RNA

Synthetase Recognition Determinants of E. coli Valine Transfer RNA

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study

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