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Smita Patel

M.Sc., IIT Bombay, India
Ph.D., Tufts University
RWJMS-Research Building/SPH 283
683 Hoes Lane
Piscataway, NJ 08854-0009

Telephone: 732-235-3372
Facsimile: 732-235-4783

Patel CV

Structure-function and dynamics of enzyme-catalyzed processes involved in genome replication and transcription.

Research Interests
The research in my laboratory is focused on understanding the molecular mechanisms of enzyme catalyzed processes of genome replication and transcription. We take a multidisciplinary approach in understanding enzymatic mechanisms. Emphasis is on the use of transient state kinetics (rapid chemical quench-flow and stopped-flow) to decipher the kinetic pathways, structural studies and mutational studies to understand structure-function, equilibrium measurements to define the thermodynamics of these processes. We are currently investigating the enzymology of a) Helicases (viral helicases, both DNA and RNA helicases, that are involved in genome replication) b) Mechanism and regulation of Transcription

Up to 2% of the genome encodes for helicases and helicase-like proteins. A growing number of helicases are also being associated with human diseases, some of which are Xeroderma Pigmentosum, Bloom's syndrome, and Werner's syndrome characterized by premature aging. Viruses also tend to encode their own helicases, and these viral helicases are potential drug targets. Greater than 2% of the human population world-wide is infected by hepatitis C virus making this virus a major human pathogen. HCV encodes its own helicase, which we are investigating to understand its mechanism of action, substrate specificity, and structure-function–studies that will be crucial in developing strategies for antiviral agents.

Biochemical studies reveal that helicases are nucleic acid motor proteins that use the chemical energy from NTP hydrolysis to move along DNA and RNA. A class of helicases assemble into hexameric rings (see figure) 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. The HCV helicase falls into a different class and does not form a ring but our studies indicate that it functions as an oligomer. The mechanism of nucleic acid unwinding by helicases is yet unknown, and our current and future studies of helicases are focused on a variety of topics, some of which are listed below:

  • Mechanism of unidirectional translocation of helicases along nucleic acids.
  • Nucleic acid unwinding mechanism (fit all-or-none unwinding kinetics using gfit.
  • Single molecule studies to measure movement and strand separation activity
  • Studies of uncoupled helicase mutants to understand the basis of energy transduction in helicases
  • Understanding the relationship between NTP hydrolysis and translocation and DNA strand separation reactions

Mechanism and Regulation of Transcription
The control of gene expression at the level of mRNA synthesis is the focus of our second research project. We are dissecting the elementary steps of various stages of transcription including initiation, promoter clearance, and elongation. In addition, we are investigating how these steps are controlled by the sequence of the promoter and by accessory proteins.

We are studying single subunit RNA polymerases such as bacteriophage T7 and mitochondrial RNA polymerase. T7 RNA polymerase is one of the best structurally characterized proteins of this class, whose single polypeptide can specifically initiate, elongate, and terminate transcription. The transcriptional efficiency of the T7 RNA polymerase is regulated both by the sequence of its promoter and by protein-protein interactions with a regulatory protein, namely T7 lysozyme. We use T7 RNA polymerase as a model system to develop methodologies to elucidate the elementary steps of transcription initiation, promoter clearance, elongation, and termination, which appear to be highly conserved in nature. The mitochondrial RNA polymerase that transcribes the mitochondrial genome shows high homology to T7 RNA polymerase. We are investigating the role of its transcription factor in aiding initiation at specific mitochondrial promoters.

We have developed fluorescence-based methods to measure the elementary steps of transcription. We employ 2-aminopurine modified DNA promoters to measure the kinetics of DNA binding and open complex formation in real time using stopped-flow methods. Similarly, we use the radiometric rapid chemical quenched-flow methods to measure the steps of RNA synthesis occurring on the enzyme active site. Our studies are focused on dissecting the transcriptional pathway, identifying the intermediates, and measuring the kinetic and thermodynamic parameters governing each step. Our goal is also to relate the identified intermediates to available structural information. Additionally, we study transcription to understand how it is controlled. These studies will form the basis to investigate transcription in higher organisms.

Patel Laboratory

  • Doyel Sen
  • Gayatri Patel
  • Manjula Pandey
  • Guo-Qing Tang
  • Vaishnavi Rajagopal
  • Anand Ramanathan
  • Swaroopa Paratkar
  • Aishwarya Despande
  • Divya Nandakumar