Structural Studies of Model RNA Helices with Relevance to Aminoacyl-tRNA Synthetase Specificity and HIV Reverse Transcription

Abstract

We describe high-resolution crystal structures of synthetic nucleic-acid fragments determined as part of an effort to understand determinants of sequence-specific protein binding on the level of double-helix structure. In a first set of experiments, 7-base-pair RNA duplexes representing the acceptor-stem helix of Escherichia coli tRNA^Ala and variants thereof were characterized at atomic resolution. The structures revealed a standard A-form double helix locally perturbed by a G·U wobble base pair at sequence position 3/70 of the tRNA. The G·U pair shows a characteristic hydration pattern which must be considered an integral part of the double-helix structure. It does not seem to exert a global effect on the duplex structure. A second experiment concerned the chimeric DNA-RNA hybrid structure formed transiently during initiation of minus-strand synthesis by the reverse transcriptase of HIV-1. The crystal structure of an 8-base-pair duplex with an RNA template strand derived from HIV-1 and a complementary strand representing the junction between the tRNA^Lys,3 RNA primer and the newly synthesized DNA strand was solved at a resolution of 1.9 Å. As before, the double helix was found to adopt standard A-type conformation with only local variations of backbone conformation. Based on the global helix structure as present in the crystal, it remains difficult to explain the preference of the reverse-transcriptase-associated RNAse H activity for certain sites of the template strand. Structural plasticity near the main cleavage site in suggested to govern cutting preferences. In both systems investigated, structural studies by NMR spectroscopy were carried out by others in parallel. In both cases, the solution structures are in partial disagreement with the crystallographic results by describing a significantly higher level of deviation from the canonical A-conformation.