Research

Crystallography of the Viral RNA Polymerase

The virus-encoded DNA-directed RNA polymerase is a novel, multifunctional, and yet a simple enzyme composed of only four equimolar subunits that include 5' (capping) and 3' (polyadenylating) activities and sufficient for basal in vitro transcription.  In addition, no other factors are required to specifically recognize and bind to late viral promoters.  Baculoviruses are the only nuclear-replicating viruses that encode their DNA-directed RNA polymerase and thus, they lend themselves as a simple eukaryotic model system to study these complex multisubunit enzymes.  The largest subunit of the baculovirus RNA polymerase, late expression factor-8 (LEF-8), has an evolutionarily conserved putative catalytic site shared by a number of enzymes across kingdoms.  The motif is found in the ß or ß' subunit of DNA-directed RNA polymerases in eubacteria and archaebacteria, chloroplasts, Drosophila (RNA polymerase II and III), Saccharomyces (RNA polymerase I, II, and III), and humans (RNA polymerase II).  Also, this motif is required in functional assays.  We are overexpressing and purifying the LEF-8 polypeptide in order to derive its crystal structure in collaboration with Dr. Ernst Schönbrunn at The University of Kansas.  The structure of the baculovirus RNA polymerase and its subunits will be instrumental for drawing structural and functional comparisons between this viral and other eukaryotic and prokaryotic RNA polymerases that share similar domains.