Philip C. Bevilacqua

RNA Folding and Catalysis

The Bevilacqua lab is interested in the folding and catalysis of ribonucleic acid (RNA), and its interactions with proteins. RNA-protein complexes carry out structural and functional roles central to the execution and regulation of many biological processes. Our laboratory focuses on biologically important systems including viral replication and the human viral response. The laboratory is problem based and uses a variety of experimental approaches including rapid mixing kinetics, fluorescence spectroscopy, UV melting, site-directed mutagenesis, combinatorial selection of RNA (or SELEX) and NMR.

Characterization of RNA catalysis and folding

The hepatitis delta virus (HDV) is a human pathogen that utilizes a catalytic RNA, or ribozyme, in its replication cycle. We are investigating fundamental catalytic and folding processes of the ribozyme. Mechanistically, we are interested in the role of RNA nucleotides as general acids and bases in the cleavage mechanism. Recent studies in our lab implicate C75 as a general acid in the cleavage mechanism (see figure). Current efforts are focused on methods for determining pKa values of critical residues, and examining the effect of individual functional groups and microenvironment on pKa perturbation. In terms of the folding mechanism, we are interested in the role of nucleotides flanking the ribozyme in overall folding. We are developing optical approaches to monitor the folded state of individual nucleotides during folding. Modified nucleosides that have favorable fluorescence properties, along with kinetic mixing experiments, are being used. These results may lead to better oligonucleotide therapeutics, and help us to understand the role of RNA in evolution.

Combinatorial approach to RNA/DNA thermodynamics

The large amount of nucleic acid sequence being determined by genome projects has generated a great interest in the computer prediction of RNA structure. RNA structure prediction is roughly 70 percent accurate, indicating room for substantial improvement. Current prediction methods require accurate thermodynamic parameters for a wide range of secondary and tertiary structural motifs including bulges; internal, hairpin, and multibranch loops; interactions among these loops; and base triples. Very few of these parameters have been determined, however. The major obstacle has been that the number of sequence combinations for a given structural element is so large that it is unrealistic to prepare and test them all. We have developed a combinatorial method to select thermodynamically stable, complex RNA structural elements from a randomized library. RNAs of different stability are separated by temperature-gradient gel electrophoresis (TGGE), excised from the gel and their sequences determined by cloning. Thermodynamic parameters are determined and used to improve the prediction of RNA structure. Novel motifs are being characterized structurally. Functional group substitution is performed to identify atoms critical to stability, and structures are probed by NMR. These experiments are being extended to the identification of stable DNA motifs such as occur in recombination and replication intermediates, ssDNA-containing viruses, and DNA enzymes.

Characterization of the RNA-dependent regulation of human viral response

The human double-stranded-RNA-activated protein kinase (PKR) is a 551 residue RNA-binding protein that contains two N-terminal copies of a conserved motif, the double-stranded RNA binding motif (dsRBM), and a C-terminal kinase domain. PKR is present in higher eukaryotes, including humans, and mediates an interferon-induced viral response. We would like to determine the rigorous kinetic mechanism for assembly of an activated PKR complex on dsRNA. PKR binding and conformational changes will be detected by monitoring the intrinsic fluorescence emission of three tryptophans located in the kinase domain and by fluorescently tagged dsRNA. Many viruses have evolved strategies for down-regulating PKR. Once the detailed mechanism for PKR activation is established, we will examine the strategies viral RNAs use to regulate this mechanism. Other issues of interest with PKR include assigning the role of the multiple dsRBMs, and identifying and determining the structure of non-dsRNA sequences involved in regulating PKR activity.