How ribonucleic acid (RNA) molecules fold into tertiary structures, how proteins recognize specific RNA structures, and how protein binding is involved in RNA functions are central questions to a number of biological processes. The broad goal of this proposal is to elucidate the high-resolution structures and mechanistic principles that underlie the formation of a particular RNA structural fold (the loop-loop """"""""kissing"""""""" motif) in three different and unique systems using multi- dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy and other biophysical methods. RNA loop-loop """"""""kissing"""""""" interactions form principally via base-pairing between loops and/or bulges with complementary nucleotide sequences and are the critical intermediate structures involved in the antisense regulation of a variety of cellular processes in bacteria. These interactions are also involved in the proper folding of certain mRNA, rRNA and catalytic RNA structures. As an important element in dimerization of genomic retroviral RNA, the loop-loop """"""""kissing"""""""" structure is also a viable target for anti-viral drugs.
The specific aims of this research are: 1) to determine the structural basis for specific interaction in two novel RNA loop-loop """"""""kissing"""""""" complexes: a homodimeric loop-loop """"""""kissing"""""""" complex formed by an RNA stem-loop derived from the dimerization initiation site (DIS) in HIV-1 genomic RNA and a loop-loop """"""""kissing"""""""" complex formed by RNA stem-loops derived from the mRNA CopA and antisense CopT repressor transcripts involved in R1 plasmid replication control; and 2) to determine the structural basis for the specific recognition of a loop- loop """"""""kissing"""""""" complex derived from the ColE1 replication control system by the protein Rom. NMR spectroscopic structural and dynamical studies will be complemented by stop-flow kinetic, thermal melting and gel electrophoresis experiments designed to assay thermodynamic stability and association/dissociation kinetics of wild type and mutant loop-loop """"""""kissing"""""""" complexes. New NMR methods, specifically applicable to 13C, 15N and/or 2H labeled RNA and RNA-protein complexes of sizes (10 kDa - 26 kDa), will also be designed which will provide higher precision and sensitivity than available with existing technology and which will access global long-range structural restraints currently unavailable for RNA molecules.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059107-05
Application #
6636281
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
1999-05-01
Project End
2005-05-31
Budget Start
2003-09-01
Budget End
2005-05-31
Support Year
5
Fiscal Year
2003
Total Cost
$178,077
Indirect Cost
Name
University of MD Biotechnology Institute
Department
Type
Organized Research Units
DUNS #
603819210
City
Baltimore
State
MD
Country
United States
Zip Code
21202
Brinson, Robert G; Miller, Jennifer T; Kahn, Jason D et al. (2016) Applying Thymine Isostere 2,4-Difluoro-5-Methylbenzene as a NMR Assignment Tool and Probe of Homopyrimidine/Homopurine Tract Structural Dynamics. Methods Enzymol 566:89-110
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Fabris, Daniele; Marino, John P; Le Grice, Stuart F J (2009) Revisiting plus-strand DNA synthesis in retroviruses and long terminal repeat retrotransposons: dynamics of enzyme: substrate interactions. Viruses 1:657-77
Brinson, Robert G; Turner, Kevin B; Yi-Brunozzi, Hye Young et al. (2009) Probing anomalous structural features in polypurine tract-containing RNA-DNA hybrids with neomycin B. Biochemistry 48:6988-97
Lee, Hui-Wen; Briggs, Katharine T; Marino, John P (2009) Dissecting structural transitions in the HIV-1 dimerization initiation site RNA using 2-aminopurine fluorescence. Methods 49:118-27
Turner, Kevin B; Yi-Brunozzi, Hye Young; Brinson, Robert G et al. (2009) SHAMS: combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids. RNA 15:1605-13
Yi-Brunozzi, Hye Young; Brinson, Robert G; Brabazon, Danielle M et al. (2008) High-resolution NMR analysis of the conformations of native and base analog substituted retroviral and LTR-retrotransposon PPT primers. Chem Biol 15:254-62
Turner, Kevin B; Brinson, Robert G; Yi-Brunozzi, Hye Young et al. (2008) Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands. Nucleic Acids Res 36:2799-810
Struble, E B; Ladner, J E; Brabazon, D M et al. (2008) New crystal structures of ColE1 Rom and variants resulting from mutation of a surface exposed residue: Implications for RNA-recognition. Proteins 72:761-8
Zhao, Chang; Marino, John P (2007) Synthesis of HIV-1 Psi-site RNA sequences with site specific incorporation of the fluorescent base analog 2-aminopurine. Tetrahedron 63:3575-3584

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