RNA



X-ray crystallographic observation of “in-line” and “adjacent” conformations in a bulged self-cleaving RNA/DNA hybrid


VALENTINA  TERESHKO a1, SCOT T.  WALLACE a2, NASSIM  USMAN a3, FRANCINE E.  WINCOTT a3 and MARTIN  EGLI a1c1
a1 Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA
a2 Department of Molecular Biology, Intercell G.m.b.H., A-1030 Vienna, Austria
a3 Department of Chemistry and Biochemistry, Ribozyme Pharmaceuticals Inc., Boulder, Colorado 80301, USA

Abstract

The RNA strand in an RNA/DNA duplex with unpaired ribonucleotides can undergo self-cleavage at bulge sites in the presence of a variety of divalent metal ions (Hüsken et al., Biochemistry, 1996, 35:16591–16600). Transesterification proceeds via an in-line mechanism, with the 2′-OH of the bulged nucleotide attacking the 3′-adjacent phosphate group. The site-specificity of the reaction is most likely a consequence of the greater local conformational freedom of the RNA backbone in the bulge region. A standard A-form backbone geometry prohibits formation of an in-line arrangement between 2′-oxygen and phosphate. However, the backbone in the region of an unpaired nucleotide appears to be conducive to an in-line approach. Therefore, the bulge-mediated phosphoryl transfer reaction represents one of the simplest RNA self-cleavage systems. Here we focus on the conformational features of the RNA that underlie site-specific cleavage. The structures of an RNA/DNA duplex with single ribo-adenosyl bulges were analyzed in two crystal forms, permitting observation of 10 individual conformations of the RNA bulge moiety. The bulge geometries cover a range of relative arrangements between the 2′-oxygen of the bulged nucleotide and the P-O5′ bond (including adjacent and near in-line) and give a detailed picture of the conformational changes necessary to line up the 2′-OH nucleophile and scissile bond. Although metal ions are of crucial importance in the catalysis of analogous cleavage reactions by ribozymes, it is clear that local strain or conformational flexibility in the RNA also affect cleavage selectivity and rate (Soukup & Breaker, RNA, 1999, 5:1308–1325). The geometries of the RNA bulges frozen out in the crystals provide snapshots along the reaction pathway prior to the transition state of the phosphoryl transfer reaction.

(Received September 22 2000)
(Revised October 23 2000)
(Accepted November 28 2000)


Key Words: DNA; hydrogen bonding; nucleotide bulge; phosphoryl transfer; ribozyme; RNA; X-ray crystallography.

Correspondence:
c1 Reprint requests to: Martin Egli, Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA; e-mail: martin.egli@vanderbilt.edu.