Investigation of Overhauser effects between pseudouridine and water protons in RNA helices

AUTOR(ES)

Newby, Meredith I.

FONTE

The National Academy of Sciences

RESUMO

The inherent chemical properties of RNA molecules are expanded by posttranscriptional modification of specific nucleotides. Pseudouridine (ψ), the most abundant of the modified bases, features an additional imino group, NH1, as compared with uridine. When ψ forms a Watson–Crick base pair with adenine in an RNA helix, NH1 is positioned within the major groove. The presence of ψ often increases thermal stability of the helix or loop in which it is found [Hall, K. B. & McLaughlin, L. (1992) Nucleic Acids Res. 20, 1883–1889]. X-ray crystal structures of transfer RNAs [e.g., Arnez, J. & Steitz, T. (1994) Biochemistry 33, 7560–7567] have depicted water molecules bridging ψNH1 groups and nearby phosphate oxygen atoms, but direct evidence for this interaction in solution has not been acquired. Toward this end, we have used a rotating-frame Overhauser effect spectroscopy-type NMR pulse sequence with a CLEAN chemical-exchange spectroscopy spin-lock pulse train [Hwang, T.-L., Mori, S., Shaka, A. J. & van Zijl, P. C. M. (1997) J. Am. Chem. Soc. 119, 6203–6204] to test for ψNH1–water cross-relaxation effects within two RNA helices: (i) a complementary duplex, in which ψ is not associated with structural change, and (ii) an RNA duplex representing the eukaryotic pre-mRNA branch-site helix from Saccharomyces cerevisiae, in which a conserved ψ extrudes the branch-site adenosine from the helix. Our data implicate a water–ψNH1 hydrogen bond both in stabilizing the complementary helix and in favoring formation of the unique structure of the branch-site helix.

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