HELICAL RNA, RNA:DNA HYBRIDS AND DNA-RNA JUNCTIONS

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RNA duplexes, RNA:DNA hybrid duplexes and DNA-RNA duplex junctions are involved in many important steps in gene expression including replication, transcription and translational control. Relatively little is known about the structure of these biologically important duplexes, largely due to the difficulty of synthesizing the large amounts of defined sequence RNA molecules required for detailed structural studies (NMR, x-ray crystallography). We have circumvented this bottleneck and have recently synthesized 20-50 mg quantities of RNA chains with defined sequences. We now propose to carry out extensive high-resolution NMR studies on the solution structure of r:r duplexes and to compare their structure to d:d duplexes of the same Studies will also be carried out on d:r hybrid duplexes and d-r-d:d-r-d chimeric duplexes containing A-B junctions. Initial insights into structure will be obtained from complete proton assignments, chemical shift changes, and interpretation of changes in coupling constants. More detailed later structural studies will involve time-dependent NOESY studies, interproton distance measurements and ab initio generation of three-dimensional structures consistent with these distances using distance geometry algorithms. The results should shed a great deal of light on the peculiarities of RNA structure, the control of transcription of DNA information into RNA information (and vice-versa) and the apparent need for RNA primers at replication forks and in the initiation of DNA replication.