The NMR structure of 31mer RNA domain of Escherichia coli RNase P RNA using its non-uniformly deuterium labelled counterpart [the 'NMR-window' concept]

The NMR structure of a 31mer RNA constituting a functionally important domain of the catalytic RNase P RNA from Escherichia con is reported. Severe spectral overlaps of the proton resonances in the natural 31mer RNA (1) were successfully tackled by unique spectral simplifications found in the partially-deuterated 31mer RNA analogue (2) incorporating deuterated cytidines [C5 (>95 atom % H-2), C2'(>97 atom % 2H)I C3' (>97 atom % H-2), C4, (>65 atom % H-2) and C5'(>97 atom % H-2)] [for the 'NMR-window' concept see: Foldesi,A. et al.(1992) Tetrahedron, 48, 9033; Foldesi,A. et al (1993) J. Biochem, Biophys. Methods, 26, 1; Yamakage,S.-l, et al. (1993) Nucleic Acids Res., 21, 5005; Agback,P. ef a. (1994) Nucleic Acids Res., 22,1404; Foldesi,A. at al. (1995) Tetrahedron, 51, 10065; Foldesi,A. et al. (1996) Nucleic Acids Res.,:24, 1187-1194]. 175 resonances have been assigned out of total of 235 non-exchangeable proton resonances in (1) in an unprecedented manner in the absence of C-13 and N-15 labelling,41 out of 175 assigned resonances could be accomplished with the help of the deuterated analogue (2). The two stems in 31mer RNA adopt an A-type RNA conformation and the base-stacking continues from stem I into the beginning of the loop I, Long distance cross-strand NOEs showed a structured conformation at the junction between stem I and loop I, The loop I-stem II function is less ordered and shows structural perturbation at and around the G11 . C22 base pair.