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

Abstract

The NMR structure of a 31mer RNA constituting a functionally important domain of the catalytic RNase P RNA from Escherichia coli 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 31 mer RNA analogue (2) incorporating deuterated cytidines [C5 (>95 atom % 2H), C2' (>97 atom % 2H), C3' (>97 atom % 2H), C4' (>65 atom % 2H) and C5' (>97 atom % 2H)] [for the 'NMR-window' concept see: Földesi,A. et al. (1992) Tetrahedron, 48, 9033; Foldesi,A. et al. (1993) J. Biochem. Biophys. Methods, 26, 1; Yamakage,S.-I. et al. (1993) Nucleic Acids Res., 21, 5005; Agback,P. et al. (1994) Nucleic Acids Res., 22, 1404; Földesi,A. et al. (1995) Tetrahedron, 51, 10065; Földesi,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 13C and 15N 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 junction is less ordered and shows structural perturbation at and around the G11 -C22 base pair.

Residues inEscherichia coliRNase P RNA Important for Cleavage Site Selection and Divalent Metal Ion Binding

“…The combined data suggest that, in particular, residues G292 and G293 in M1 RNA are exposed and accessible for base-paring with the substrate and are not involved in conventional Watson-Crick interactions with other parts of M1 RNA; for example, nucleotides in the upper part of the internal loop. This is also supported by our studies in which the structure of an RNA representing the P7-loop was determined by NMR spectroscopy (Glemarec et al, 1996). Thus, we conclude that the structural integrity of the P7-loop is important in making the conserved GGU motif accessible for base-pairing (see also below).…”

Structural plasticity and Mg2+ binding properties of RNase P P4 from combined analysis of NMR residual dipolar couplings and motionally decoupled spin relaxation

“…Chemical shift mapping data were available in certain cases without corresponding K d values because titration spectra were recorded with reduced resolution in the indirect dimension. (C) Electrostatic surface potential of P4 computed using the B-type X-ray structure (Glemarec et al 1996;Kazantsev et al 2005) with negative and positive potentials indicated in red and blue, respectively.…”

Eukaryotic Ribonuclease P: A Plurality of Ribonucleoprotein Enzymes

“…In the ribozyme reaction, the P15 loop has been shown to base pair with the 3′-CCA sequence of the substrate pre-tRNA (and product) (28,120). Kinetic and biophysical analysis suggested that there might be a metal ionbinding site in the bacterial P15 loop (121)(122)(123). However, this interaction may be less important in the cleavage catalyzed by the bacterial holoenzyme (123).…”