The research in my laboratory is focused on understanding the functions and mechanisms of enzymes involved in genome replication and transcription. We use among various methods transient state kinetics (rapid chemical quench-flow and stopped-flow) and fluorescence including time-resolved FRET to study enzymatic reaction dynamics. We combine enzymological studies with structure-function and single molecule studies in collaboration to achieve a detailed understanding of the workings of an enzyme. We use powerful computational methods to model the experimental data to understand and decipher the detailed pathways of enzymatic reactions. We are currently investigating a) replicative enzymes: helicases and DNA polymerase b) transcriptional enzymes: DNA-dependent RNA polymerase. and c) viral replicases: HCV RNA helicase and RNA polymerase.
Helicases are ubiquitous proteins that catalyze the separation of duplex DNA strands, the removal of secondary structures in RNA, and the reorganization of proteins bound to DNA. Being involved in almost all DNA and RNA metabolism processes, helicases and related proteins constitute greater than 2% of the genome. Recent findings show that defects in helicases lead to human diseases such as cancer and premature aging.
We are investigating several helicases that function in DNA and RNA replication and in transcription termination. Biochemical studies reveal that helicases are nucleic acid motor proteins that use the chemical energy from NTP hydrolysis to move along DNA and RNA. Understanding the coupling between NTP hydrolysis and mechanics of movement along DNA or RNA is an important goal of our research. A class of helicases assembles into hexamer rings including T7 DNA helicase and the Rho transcription termination factor. These ring helicases bind single stranded nucleic acid through their central channel and their subunits display a high degree of cooperativity, which is among the topic of our studies.
Many viruses encode their own helicases that are potential antiviral targets. Hepatitis C virus has infected greater than 2% of the human population world-wide making this virus a major human pathogen. We study the HCV proteins including the helicase and replicase to understand their function and mechanism of action, substrate specificity, and structure-function-studies to ultimately aid in obtaining agents to inhibit their function.
Mechanism and Regulation of Transcription
Understanding gene expression at the level of mRNA synthesis is the focus of our second research project. We are dissecting the elementary steps of the various stages of transcription including initiation, promoter clearance, and elongation using transient state kinetic methods. We are investigating how these steps are controlled by the sequence of the promoter and by accessory proteins to obtain a quantitative understanding of gene expression.
The RNA polymerases encoded by certain bacteriophages have a simple and economical organization, Interestingly, these polymerases show homology to mitochondrial and chloroplast RNA polymerases. T7 RNA polymerase is one of the best structurally characterized proteins of this class. We use T7 RNA