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 structural and mechanistic principles that underlie the formation of a particular RNA structural fold, the loop-loop kissing motif, using multi-dimensional heteronuclear nuclear magnetic resonance (NMR) and fluorescence spectroscopy as the primary tools. RNA loop-loop kissing interactions form principally via base-pairing between loops and/or bulges with complementary nucleotide sequences and are essential structures involved in a variety of cellular processes in bacteria, in the dimerization of retroviral genomic RNA and in the proper folding of certain messenger and catalytic RNAs. Understanding how RNA kissing interactions function at the molecular level requires detailed studies of the structure, dynamics and protein interactions of these complexes. The proposed studies will focus on two systems in which RNA kissing interactions are observed to nucleate RNA dimerization and specifically direct further RNA refolding through stem strand invasion and helix exchange: the HIV-1 dimerization initiation site (DIS) genomic dimer linkage and the antisense regulation of R1 plasmid repA mRNA translation. The role of the auxiliary proteins HIV-1 nucleocapsid (NCp7) and Gag in chaperoning DIS kissing dimer refolding will also be examined. Specifically, our research will aim to: 1) Examine the role of RNA stem-loop structure in determining the directionality and extent of helix exchange in the repA mRNA-repressor RNA kissing interaction; 2) Examine the role of RNA kissing dimer structure and dynamics in NCp7 chaperoned HIV-1 DIS maturation; 3) Investigate dimerization and maturation of DIS within the context of HIV-1 leader sequences using segmental labeling and fluorescence/NMR spectroscopy; 4) Analyze NCp7 and Gag chaperoned maturation of the DIS kissing dimer and screen for inhibitors of this process. Overall, it is anticipated that these studies will aid in the design of more effective antisense therapies, as well as provide rational approaches for designing inhibitors against HIV-1 DIS, NCp7 and/or Gag that could facilitate development of new antiviral therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059107-07
Application #
7065602
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
1999-05-01
Project End
2009-05-31
Budget Start
2006-06-01
Budget End
2007-05-31
Support Year
7
Fiscal Year
2006
Total Cost
$236,850
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
Aduri, Raviprasad; Briggs, Katharine T; Gorelick, Robert J et al. (2013) Molecular determinants of HIV-1 NCp7 chaperone activity in maturation of the HIV-1 dimerization initiation site. Nucleic Acids Res 41:2565-80
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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