The broad objective of this work is to develop structural and chemical principles which make it possible to find highly specific ways to interfere with fundamental biological processes in the propagation of the HIV virus. Specific projects include the use of NMR methods for determination of the structure of peptide fragments of the HIV transactivating (Tat) protein with its RNA target, TAR. Building on our earlier demonstration of a long- lived and specific complex between a 24-amino acid peptide and a 27- nucleotide segment of TAR (deltaTAR), we will explore intensively the structure of this complex. Comparative studies will also be undertaken in which mutant deltaTAR RNAs with much reduced complex stability and lifetime are substituted for the wild type, and in which smaller peptide fragments containing the basic region of Tat are in complex with wild type deltaTAR. Stopped flow methods will be used to probe the possible role of the kinetics of peptide and RNA conformational changes in limiting the rate of complex formation. A second objective will be to assay for physical interaction between Tat bound to TAR and regulatory proteins, specifically Sp1, bound to promoter elements. Specially constructed bent DNA molecules which bring the Tat-TAR element into apposition with an Sp1 binding site will be used for this purpose. Finally, we will initiate studies of the ability of the enediyne class of compounds to modify or cleave viral proteins, specifically gp120. Preliminary work shows that these compounds are able to react with proteins, including apparent dimerization and cleavage events. Strategies will be developed for targeting cleavage reactions to the region of gp120 which is responsible for CD4 binding.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM039546-10
Application #
6296697
Study Section
Project Start
1996-09-01
Project End
1999-08-31
Budget Start
1996-10-01
Budget End
1997-09-30
Support Year
10
Fiscal Year
1996
Total Cost
Indirect Cost
Name
Yale University
Department
Type
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Riley, Kasandra J; Steitz, Joan A (2013) The ""Observer Effect"" in genome-wide surveys of protein-RNA interactions. Mol Cell 49:601-4
Brautigam, C A; Aschheim, K; Steitz, T A (1999) Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment. Chem Biol 6:901-8
Ota, N; Stroupe, C; Ferreira-da-Silva, J M et al. (1999) Non-Boltzmann thermodynamic integration (NBTI) for macromolecular systems: relative free energy of binding of trypsin to benzamidine and benzylamine. Proteins 37:641-53
Friedman, J M (1999) Interconversion between 3D molecular representations: some macromolecular applications of spherical harmonic-Bessel expansions about an arbitrary center. Comput Chem 23:9-23
Ippolito, J A; Steitz, T A (1998) A 1.3-A resolution crystal structure of the HIV-1 trans-activation response region RNA stem reveals a metal ion-dependent bulge conformation. Proc Natl Acad Sci U S A 95:9819-24
Jaeger, J; Restle, T; Steitz, T A (1998) The structure of HIV-1 reverse transcriptase complexed with an RNA pseudoknot inhibitor. EMBO J 17:4535-42
Brautigam, C A; Steitz, T A (1998) Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates. J Mol Biol 277:363-77
Friedman, J M (1997) Fourier-filtered van der Waals contact surfaces: accurate ligand shapes from protein structures. Protein Eng 10:851-63
Mishima, Y; Steitz, J A (1995) Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to reverse transcriptase from human immunodeficiency virus type I. EMBO J 14:2679-87
Long, K S; Crothers, D M (1995) Interaction of human immunodeficiency virus type 1 Tat-derived peptides with TAR RNA. Biochemistry 34:8885-95

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