The solution structure of a RNA pentadecamer comprising the anticodon loop and stem of yeast tRNAPhe. A 500 MHz 1H-n.m.r. study

Abstract

A 500 MHz 1H-NMR study on the semi-synthetic RNA pentadecamer 5''-r(C-A-G-A-Cm-U-Gm-A-A-Y-A-.PSI.-m5C-U-G) [Cm = 2''-O-methylcytidine, Gm = 2''-O-methylguanosine, Y = wybutine, .PSI. = pseudouridine, M5C = 5-methyl cytidine] comprising the anticodon loop and stem (residues 28-42) of [baker''s] yest tRNAPhe is presented. By using pre-steady-state nuclear-Overhauser-effect measurements all exchangeable and non-exchangeable base proton resonances, all H1'' ribose resonances and all methyl proton resonances are assigned and over 70 intra- and inter-nucleotide interproton distances determined. From the distance data the solution structure of the pentadecamer is solved by model-building. The pentadecamer adopts a hairpin-loop structure in solution with the loop in a 3''-stacked conformation. This structure is both qualitatively and quantitatively remarkably similar to that of the anticodon loop and stem found in the crystal structures of tRNAPhe with an overall root-mean-square difference of 0.12 nm between the interproton distances determined by NMR and X-ray crystallography. The hairpin-loop solution structure of the pentadecamer is very stable with a melting temperature of 53.degree. C in 500 mM-KCl, and the structural features responsible for this high stability are discussed. Interaction of the pentadecamer with the ribotrinucleoside diphosphate UpUpC, one of the codons for the amino acid phenylalanine, results only in minor perturbations in the structure of the pentadecamer, and the 3''-stacked conformation of the loop is preserved. The stability of the pentadecamer-UpUpC complex (K .apprx. 2.5 .times. 104 M-1 at 0.degree. C) is approximately an order of magnitude greater than that of the tRNAPhe-UpUpC complex.