The broad long-term objective of this work is to characterize the local structural and dynamical features of RNA molecules, particularly those which regulate of biological function. This is consistent with the key hypothesis of Structural Biology that the three-dimensional arrangements of biomolecules are intimately related to their function. There is also the implicit understanding that structural change, expressed rigorously in thermodynamic and kinetic terms, is vital to understanding biological processes. During this project period we will use NMR to characterize specific RNA substructural units, and and the interactions of these substructures in larger, biologically relevant assemblies. The primary biological targets planned for this project are the binding site for the coat protein of R17 virus, and substructures from the 5 prime-leader sequence of HIV-1 involved in the packaging signal for the virus. Our primary aims for this project period are to: (1) Refine the ~wild- type~ variant of the structure of a 24-nucleotide R17 viral RNA using NMR distance and angle constraints. (2) Synthesize and use NMR to analyze RNA hairpins corresponding to substructures of the packaging signal in the HIV-1 genome. Hypothesis for their 3D folding will be tested by constraining their structures with very stable base-pairing blocks in the stems. (3) Use quantitative footprinting to establish the 2D folded structures of HIV-1 leader hairpins, both individually and as a part of larger superstructures. (4) Specifically label segments of the HIV-1 packaging signal with 13Carbon and 15N, and begin the process of determining its 3D structure. The whole HIV packaging signal may comprise 120nt. Even with full 13C and 15N labeling the spectrum would be difficult to interpret. Labeled hairpins will be ligated into unlabeled versions of the whole packaging signal. The key targets of our studies are subdomains of RNA viruses that known to participate in the control of processes used by viruses to package the RNA genome into infectious particles. An understanding of the structure may provide tools for designing new antiviral strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032691-12
Application #
2900582
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1983-03-01
Project End
2001-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
12
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Syracuse University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
002257350
City
Syracuse
State
NY
Country
United States
Zip Code
13244
Ouyang, Wei; Okaine, Stephen; McPike, Mark P et al. (2013) Probing the RNA binding surface of the HIV-1 nucleocapsid protein by site-directed mutagenesis. Biochemistry 52:3358-68
Mohammad, Mohammad M; Iyer, Raghuvaran; Howard, Khalil R et al. (2012) Engineering a rigid protein tunnel for biomolecular detection. J Am Chem Soc 134:9521-31
Athavale, Shreyas S; Ouyang, Wei; McPike, Mark P et al. (2010) Effects of the nature and concentration of salt on the interaction of the HIV-1 nucleocapsid protein with SL3 RNA. Biochemistry 49:3525-33
DeCiantis, Christopher L; Jensen, Danielle K; Hudson, Bruce S et al. (2007) A nucleic acid switch triggered by the HIV-1 nucleocapsid protein. Biochemistry 46:9164-73
Rivera, Sharon A; Hudson, Bruce S (2006) Rapid exchange luminescence: nitroxide quenching and implications for sensor applications. J Am Chem Soc 128:18-9
McPike, Mark P; Goodisman, Jerry; Dabrowiak, James C (2004) Specificity of neomycin analogues bound to the packaging region of human immunodeficiency virus type 1 RNA. Bioorg Med Chem 12:1835-43
Cavaluzzi, Michael J; Borer, Philip N (2004) Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA. Nucleic Acids Res 32:e13
Yuan, YiQiong; Kerwood, Deborah J; Paoletti, Andrew C et al. (2003) Stem of SL1 RNA in HIV-1: structure and nucleocapsid protein binding for a 1 x 3 internal loop. Biochemistry 42:5259-69
McPike, Mark P; Goodisman, Jerry; Dabrowiak, James C (2002) Footprinting and circular dichroism studies on paromomycin binding to the packaging region of human immunodeficiency virus type-1. Bioorg Med Chem 10:3663-72
Shubsda, Michael F; Paoletti, Andrew C; Hudson, Bruce S et al. (2002) Affinities of packaging domain loops in HIV-1 RNA for the nucleocapsid protein. Biochemistry 41:5276-82

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