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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042896-04
Application #
3301827
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1989-07-01
Project End
1994-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
4
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
Schools of Arts and Sciences
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Salazar, M; Fedoroff, O Y; Reid, B R (1996) Structure of chimeric duplex junctions: solution conformation of the retroviral Okazaki-like fragment r(ccca)d(AATGA).d(TCATTTGGG) from Moloney murine leukemia virus. Biochemistry 35:8126-35
Fedoroff OYu; Salazar, M; Reid, B R (1996) Structural variation among retroviral primer-DNA junctions: solution structure of the HIV-1 (-)-strand Okazaki fragment r(gcca)d(CTGC).d(GCAGTGGC). Biochemistry 35:11070-80
Zhu, L; Reid, B R (1995) An improved NOESY simulation program for partially relaxed spectra: BIRDER. J Magn Reson B 106:227-35
Zhu, L; Salazar, M; Reid, B R (1995) DNA duplexes flanked by hybrid duplexes: the solution structure of chimeric junctions in [r(cgcg)d(TATACGCG)]2. Biochemistry 34:2372-80
Salazar, M; Fedoroff OYu; Zhu, L et al. (1994) The solution structure of the r(gcg)d(TATACCC):d(GGGTATACGC) Okazaki fragment contains two distinct duplex morphologies connected by a junction. J Mol Biol 241:440-55
Wang, A C; Kennedy, M A; Reid, B R et al. (1994) A solid-state 2H NMR investigation of purine motion in a 12 base pair RNA duplex. J Magn Reson B 105:1-10
Fedoroff OYu; Salazar, M; Reid, B R (1993) Structure of a DNA:RNA hybrid duplex. Why RNase H does not cleave pure RNA. J Mol Biol 233:509-23
Salazar, M; Champoux, J J; Reid, B R (1993) Sugar conformations at hybrid duplex junctions in HIV-1 and Okazaki fragments. Biochemistry 32:739-44
Salazar, M; Fedoroff, O Y; Miller, J M et al. (1993) The DNA strand in DNA.RNA hybrid duplexes is neither B-form nor A-form in solution. Biochemistry 32:4207-15
Wang, A C; Kim, S G; Flynn, P F et al. (1992) Errors in RNA NOESY distance measurements in chimeric and hybrid duplexes: differences in RNA and DNA proton relaxation. Biochemistry 31:3940-6

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