Abstract

The 55-nt long RNA, modelling a three-way junction, with non-uniformly incorporated deuterated nucleotides has been synthesised in a pure form. The NMR-window part in this partially deuterated 55mer RNA consists of natural non-enriched nucleotide blocks at the three-way junction (shown in a square box in Fig.2), whereas all other nucleotides of the rest of the molecule are partially deuterated ( > 97 atom% H-2 at C2', C3', C5', C5, and similar to 50 atom% H-2 at C4'). The secondary structure of this 55mer RNA was determined by 2D H-1 NOESY spectroscopy in DO or in 10% D2O-H2O mixture. The use of deuterated building blocks in the specific region of the 55mer RNA allowed us to identify two distinct A-type RNA helices in a straightforward manner by observing connectivities of H1' with the basepaired imino and the aromatic H2 of all adenosine nucleotides as the first step for the determination of its tertiary structure in a cost- and time-effective manner without employing any C-13/N-15 labelling. These two decameric helices involve 40 nucleotides, for which all non-exchangeable H1', H6, H2, H8 and H5 protons (all 40 H1', all 40 H6 or H8 aromatics, all seven H2 of adenine nucleotide and all four non-deuterated H5 of cytosines) as well as all 16 exchangeable imino protons (with the exception of four terminal basepairs) and 16 amino protons of cytosines have been assigned. Since all aromatic-H2', H3' as well as H5'/5 " crosspeaks from partially deuterated residues have been eliminated from the NMR spectra, the observation of natural nucleotide residues in the NMR window part has essentially been simplified. It has been found that the crosspeaks from the natural nucleotides located at the three-way junction in the NMR-window part show different degrees of line-broadening, thereby indicating that the various nucleotide: residues have very different mobilities with respect to themselves as well as compared to other nucleotides in the helices. The assignment of H2' and H3' in the NMR-window part has been made based on NOESY and DQF-COSY crosspeaks. It is noteworthy that, even in this preliminary study, it has been possible to identify 10 H2' out of total 14 and 9 H3' out of 14. The data show that expanded AU containing a tract of 55mer RNA does not self-organise into a tight third helix, as the two decameric A-type helices. across the three-way junction which is evident from the absence of any additional imino protons, except those that already have been assigned fur the two decameric helices. (C) 2000 Published by Elsevier Science B.V. All rights reserved.

Keywords

H-2 labelling; 55mer RNA; NMR; structure; selective H-2 incorporation

Published in

Journal of Biochemical and Biophysical Methods
2000, Volume: 42, number: 3, pages: 153-178

UKÄ Subject classification

Biochemistry and Molecular Biology


Publication identifier

DOI: https://doi.org/10.1016/S0165-022X(99)00057-3

Permanent link to this page (URI)

https://res.slu.se/id/publ/108056