The dynamic NMR structure of the T psi C-loop: implications for the specificity of tRNA methylation.
tRNA (m5U54)-methyltransferase (RUMT) catalyzes the S-adenosylmethionine-dependent methylation of uridine-54 in the T psi C-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 T psi-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 T psi-loop in solution in conjunction with extensive binding studies of RUMT with the T psi C-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.
Yao
L J
LJ
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
James
T L
TL
Kealey
J T
JT
Santi
D V
DV
Schmitz
U
U
D001483:Base Sequence; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008745:Methylation; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000737:chemistry*; D013379:Substrate Specificity; D013816:Thermodynamics; D014158:Transcription, Genetic; D012359:tRNA Methyltransferases / Q000378:metabolism*
D012360:RNA, Transfer, Phe; D012343:RNA, Transfer; D012359:tRNA Methyltransferases; C022769:tRNA(uracil-5)-methyltransferase