The dynamic NMR structure of the TψC-loop: Implications for the specificity of tRNA methylation

Letitia J. Yao, Thomas L. James, James T. Kealey, Daniel V. Santi, Uli Schmitz

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26 Scopus citations

Abstract

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the S-adenosylmethionine-dependent methylation of uridine-54 in the TψC-loop of all transfer RNAs in E. coli to form the 54-ribosylthymine residue. However, in all tRNA structures, residue 54 is completely buried and the question arises as to how RUMT gains access to the methylation site. A 17-mer RNA hairpin consisting of nucleotides 49-65 of the Tψ-loop is a substrate for RUMT Homonuclear NMR methods in conjunction with restrained molecular dynamics (MD) methods were used to determine the solution structure of the 17-mer T-arm fragment. The loop of the hairpin exhibits enhanced flexibility which renders the conventional NMR average structure less useful compared to the more commonly found situation where a molecule exists in predominantly one major conformation. However, when resorting to softer refinement methods such as MD with time-averaged restraints, the conflicting restraints in the loop can be satisfied much better. The dynamic structure of the T-arm is represented as an ensemble of 10 time-clusters. In all of these, U54 is completely exposed. The flexibility of the Tψ-loop in solution in conjunction with extensive binding studies of RUMT with the TψC-loop and tRNA suggest that the specificity of the RUMT/ tRNA recognition is associated with tRNA tertiary structure elements. For the methylation, RUMT would simply have to break the tertiary interactions between the D- and T-loops, leading to a melting of the T-arm structure and making U54 available for methylation.

Original languageEnglish (US)
Pages (from-to)229-244
Number of pages16
JournalJournal of biomolecular NMR
Volume9
Issue number3
DOIs
StatePublished - Jan 1 1997

Keywords

  • Dynamic NMR refinement
  • MD with time-averaged restraints
  • RNA/protein recognition
  • RUMT
  • TψC-loop

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