B.S. (1999) University of South Carolina

Ph.D. (2006) University of Rochester Medical Center

Postdoctoral Fellow: HHMI at the University of MD Baltimore County

513-529-8072

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General Interests: HIV, Alternative splicing, RNA biophysical chemistry, and Protein-RNA interactions

Techniques: NMR spectroscopy, Isothermal titration calorimetry, X-ray Crystallography, and Computational methods

Research in the Tolbert group focuses on structure-function relationships involved in RNA processing. The underlying theme of our research is to develop quantitative models of RNA function that are based on high-resolution structures and biophysical principles. In all cases, our models will be tested for their biological significance. To realize these goals, our scientific approach is intrinsically collaborative and multidisciplinary incorporating aspects of molecular biology, biochemistry, physical chemistry, and structural biology. One major area of interest in the group is to understand the mechanism of alternative splice site utilization in HIV-1, the causative agent of AIDS. Alternative splicing of the HIV-1 genome is essential to virus survival and involves numerous interactions between cis RNA elements and trans host factors. Two classes of host factors, hnRNP and SR proteins, act to either suppress or stimulate alternative splicing of the HIV-1 genome, respectively. SR proteins recognize viral RNA elements known as exon splicing enhancers (ESEs) and function to recruit the Spliceosome to the 3’ intron-exon junctions. Conversely, hnRNP proteins antagonize Spliceosome assembly by binding viral RNA elements of two types: (i) intronic splicing silencers (ISSs) and (ii) exonic splicing silencers (ESSs). A fundamental aim of this research component is to establish high resolution structural models and thermodynamic profiles of hnRNP and SR proteins bound to their cognate viral RNA receptors. The contributions of this research may lead to the development of a novel class of HIV pharmaceuticals.

A second research interest of the group is to understand how environmental factors, such as divalent cations and pH, influence RNA local structure and folding. In the cell, RNA is exposed to varying solute and pH gradients. Therefore, it is advantageous to understand how pH and solute fluctuations influence RNA structure and stability. The major aim of this research component is to determine microscopic pKas for unpaired adenosines and cytosines commonly found at bulged, hairpin, and internal loop regions of RNA. Small RNA mimics of ribosomal RNA structural elements enriched in unpaired adenosines and cytosines will be studied via heteronuclear NMR spectroscopy at varying pH values. The contributions of this research will provide insight into the influence of pKa perturbations on local RNA structure. Additionally, the results may serve as benchmarks for computational chemist who are attempting to predict pKa values of RNA nucleotides located in non-canonical regions.

Publications:

Tolbert BS, Kinde B, Miyazaki Y, Stark P, Barton S, Case D, Bax A, Summers MF (2009) Method for obtaining 13C residual chemical shift anisotropies and 1H-13C residual dipolar couplings and their application to RNA structure determination. Submitted

Tolbert BS, Kennedy SD, Schroeder SJ, Krugh TR, and Turner DH (2006) The NMR structures of (rGCUGAGGCU)2 and (rGCGGAUGCU)2: Probing the structural features that shape the thermodynamic stability of GA pairs. Biochemistry 46, 1511-152

Tolbert BS, Tajc SG, Webb H, Snyder J, Nielsen J, Miller BL, Basavappa R (2005) The active site cysteine of ubiquitin-conjugating enzymes has an elevated pKa: Functional implications. Biochemistry 44, 16385-16391.

Tajc SG, Tolbert BS, Basavappa R, Miller BL (2004) Direct determination of pKa by Isothermal Titration Microcalorimetry. JACS 126, 10508-10509.

Petri E, Tolbert B, Basavappa R (2004) Structural analysis of ubiquitin conjugating Enzymes: linchpins in the ubiquitination machinery. Recent Research Development in Experimental Medicine 1, 1-17.

Basavappa R, Petri E, Tolbert B (2003) A quick and gentle method for mounting crystals in capillaries. Journal of Applied Crystallography 36, 1297-1298.