RNA STRUCTURE DETERMINATION BY DOUBLE QUANTUM COHERENCE ESR

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Studies of RNA are fundamental for the global genomics project. It plays a key role in translating genetic information stored in DNA and manifesting it via the ribosome machinery. The study of viral and bacterial RNA is an important component of RNA research and offers insights into disease control. There is a large, ongoing effort in RNA sequencing as well as efforts to obtain 3-D structures and their relation to RNA function. In our initial effort, we applied DQC ESR to measure end-to-end distances in A-form RNA. Three RNAs with 13, 16, and 26 base pairs were spin-labeled by 3-iodo-acetamido-proxyl attached to thiouridine. The 5'-5' distances were evaluated from DQC, yielding the distances between electron spins on 5-5' attached nitroxides of 40, 55, and 72¿, respectively, which agrees well with the proposed structures. A short communication describing the application of double-quantum filtered refocused primary echo (DQF-RPE) to 26 b.p. RNA is in press in J. Am. Chem. Soc. The ongoing collaboration with S.Butcher, is poised to combine NOE and DQC constraints to improve structure refinement. This will include the study of a 30 kDa RNA homodimer with a helical packing motif known as the tetraloop receptor. This motif plays an important role in tertiary structure formation in many different ribozymes. The second RNA structure of interest is derived from HIV-1 stem-loop domain involved in translational frameshifting (Staple and Butcher, NAR 2003, pdb code 1PJY). This RNA structure is essential for viral replication, but occurs in the context of a larger (45 nucleotide) RNA of unknown structure. The uncertainty in the structure is the relationship between the two helices, which are separated by a 3 nucleotide purine "hinge" region. The length of the molecule, based on molecular modeling, is approximately 71¿. However, this distance is unknown and may be less than 71¿ if the hinge region causes a bend between the two helices, as expected.