Structural studies of model RNA helices with relevance to aminoacyl-tRNA synthetase specificity and HIV reverse transcription

We describe high-resolution crystal structures of synthetic nucleic-acid fr agments determined as part of an effort to understand determinants of seque nce-specific protein binding on the level of double-helix structure. In a f irst set of experiments, 7-base-pair RNA duplexes representing the acceptor -stem helix of Escherichia coli tRNA(Ala) and variants thereof were charact erized at atomic resolution. The structures revealed a standard A-form doub le 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 whic h must be considered an integral part of the double-helix structure. It doe s not seem to exert a global effect on the duplex structure. A second exper iment concerned the chimeric DNA-RNA hybrid structure formed transiently du ring initiation of minus-strand synthesis by the reverse transcriptase of H IV-1. The crystal structure of an 8-base-pair duplex with an RNA template s trand derived from HIV-1 and a complementary strand representing the juncti on between the tRNA(Lys,3) RNA primer and the newly synthesized DNA strand was solved at a resolution of 1.9 Angstrom. 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 th e crystal, it remains difficult to explain the preference of the reverse-tr anscriptase-associated RNAse H activity for certain sites of the template s trand. Structural plasticity near the main cleavage site in suggested to go vern 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 crystallograph ic results by describing a significantly higher level of deviation from the canonical A-conformation.