CHARACTERIZATION OF THE MONOMERIC CONFORMATION OF THE HIV-1 5' LEADER

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http://hdl.handle.net/11603/15080

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UMBC Theses and Dissertations

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Author/Creator ORCID

Date

2015-01-01

Type of Work

dissertations
Text

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Chemistry & Biochemistry

Program

Biochemistry

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This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu
Distribution Rights granted to UMBC by the author.

Subjects

HIV
NMR
RNA
Structural Biology

Abstract

The structured 5' leader (5'-L) of the HIV genome regulates several important steps in the HIV life cycle and is the most highly conserved region of the genome. Previous work identified that the 5'-L exists in two mutually exclusive conformations: a monomeric and a dimeric conformation. This work focuses on enhancing our understanding of the monomeric conformation of the 5'-L, and its role in the HIV life cycle. Structural studies of the 5'-L are hindered by its large size: at 356 nucleotides it is approximately thirteen times larger than the average nuclear magnetic resonance (NMR) derived RNA structure of 27 nucleotides. To overcome these difficulties, a novel NMR strategy, long-range probing by Adenosine Interaction Detection (lr-AID), was utilized to identify the interactions in the 5'-L that stabilize the monomeric conformation. In contrast to previous predictions, we discovered that the monomeric conformation of the 5'-L is stabilized by sequestration of the dimerization initiation site (DIS) in base pairing with residues 105 through 109 of the unique 5' region. Identification of the base pairing interactions that stabilize the monomeric conformation allowed the structure of the monomeric 5'-L to be probed by NMR. Two-dimensional proton-proton NOESY spectra of predicted secondary structure elements were compared to the spectrum of the monomeric 5'-L. The presence of a number of these elements including the top of the TAR stemloop, the intact DIS stem, the splice-donor stemloop, and an extended ? stemloop were confirmed by NMR. These findings map out large portions of the monomeric 5'-L secondary structure, allowing development of a smaller construct for structural studies of a monomeric core. Additionally, inconsistencies in the reported 5' transcription start site (TSS) of the HIV genomic RNA were identified. As some of the differences were reported to result from the presence of the 5'-7-methylguanosine cap, the capped 5'-L corresponding to each TSS was synthesized and compared. Studies of the monomer-dimer equilibrium of the capped 5'-L corresponding to the different TSSs revealed that the TSS affects the dimerization propensity of the 5'-L. Our increased understanding of the structure of the monomeric 5'-L provides insights into how this region regulates important events in the HIV life cycle.