Smita Patel
Professor

UMDNJ
Department of Biochemistry
Room 836
675 Hoes Lane
Piscataway. N. J. 08854
(732) 235-3372
FAX - 4783
patelss@umdnj.edu

Dynamics of molecular motors in genome replication and transcription

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

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 polymerase as a model system to develop new methods to elucidate the elementary steps of transcription.

Selected Publications

Tang GQ, Roy R, Ha T, Patel SS. (2008) Transcription initiation in a single-subunit RNA polymerase proceeds through DNA scrunching and rotation of the N-terminal subdomains. Mol Cell. 30(5):567-77.

Rasnik I, Jeong YJ, McKinney SA, Rajagopal V, Patel SS, Ha T. (2008) Branch migration enzyme as a Brownian ratchet. EMBO J. 27(12):1727-35.

Donmez I, Patel SS. (2008) Coupling of DNA unwinding to nucleotide hydrolysis in a ring-shaped helicase. EMBO J. 27(12):1718-26.

Liu SW, Rajagopal V, Patel SS, Kiledjian M. (2008) Mechanistic and Kinetic Analysis of the DcpS Scavenger Decapping Enzyme. J Biol Chem. 283(24):16427-36.

Pandey M, Patel SS, Gabriel A. (2008) Kinetic pathway of pyrophosphorolysis by a retrotransposon reverse transcriptase. PLoS ONE. 3(1):e1389.

Rajagopal V, Patel SS. (2008) Single strand binding proteins increase the processivity of DNA unwinding by the hepatitis C virus helicase. J Mol Biol. 376(1):69-79.

Guhaniyogi J, Wu T, Patel SS, Stock AM. (2008) Interaction of CheY with the C-terminal peptide of CheZ. J Bacteriol. 190(4):1419-28.

Johnson DS, Bai L, Smith BY, Patel SS, Wang MD. (2007) Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase. Cell. 129(7):1299-309.

Bandwar RP, Ma N, Emanuel SA, Anikin M, Vassylyev DG, Patel SS, McAllister WT. (2007) The transition to an elongation complex by T7 RNA polymerase is a multistep process. J Biol Chem. 282(31):22879-86.

Donmez I, Rajagopal V, Jeong YJ, Patel SS. (2007) Nucleic acid unwinding by hepatitis C virus and bacteriophage T7 helicases is sensitive to base pair stability. J Biol Chem. 282(29):21116-23.

Singh K, Srivastava A, Patel SS, Modak MJ. (2007) Participation of the fingers subdomain of Escherichia coli DNA polymerase I in the strand displacement synthesis of DNA. J Biol Chem. 282(14):10594-604.

Picha KM, Patel SS, Mandiyan S, Koehn J, Wennogle LP. (2007) The role of the C-terminal domain of protein tyrosine phosphatase-1B in phosphatase activity and substrate binding.
J Biol Chem. 282(5):2911-7.

Anand VS, Patel SS. (2006) Transient state kinetics of transcription elongation by T7 RNA polymerase. J Biol Chem. 281(47):35677-85.

Donmez I, Patel SS.(2006) Mechanisms of a ring shaped helicase. Nucleic Acids Res. ;34(15):4216-24. Review.

Bandwar RP. Tang GQ. Patel SS. (2006) Sequential release of promoter contacts during transcription initiation to elongation transition. J Mol Biol. 360(2):466-83.

Adelman JL. Jeong YJ. Liao JC. Patel G. Kim DE. Oster G. Patel SS. (2006) Mechanochemistry of transcription termination factor Rho. Mol Cell. 22(5):611-21.

Patel SS. Donmez I. (2006) Mechanisms of helicases. J Biol Chem. May 2; [Epub ahead of print]

Tang GQ. Patel SS. (2006) Rapid binding of T7 RNA polymerase is followed by simultaneous bending and opening of the promoter DNA. Biochemistry. 45(15):4947-56.

Tang GQ. Patel SS. (2006) T7 RNA polymerase-induced bending of promoter DNA is coupled to DNA opening. Biochemistry. 45(15):4936-46.

Sims RJ 3rd. Chen CF. Santos-Rosa H. Kouzarides T. Patel SS. Reinberg D. (2005) Human but not yeast CHD1 binds directly and selectively to histone H3 methylated at lysine 4 via its tandem chromodomains. J Biol Chem. 280(51):41789-92.

Tang GQ. Bandwar RP. Patel SS. (2005) Extended upstream A-T sequence increases T7 promoter strength. J Biol Chem. 280(49):40707-13.

Stano. NM. Jeong. Y-J. Donmez. I.. Tummallapali. P. Levin MK. Patel. SS (2005) DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase. Nature. 435:370-3.

Liao. J-C. Jeong. Y-J. Kim. D-E.. Patel. SS. Oster G (2005) Mechanochemistry of T7 DNA helicase. J. Mol. Biol in press.

Levin MK. Gurjar. M. Patel SS (2005) A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase. Nat Struct Mol Biol. 5:429-35.

Jeong YJ. Levin MK. Patel SS. (2004) The DNA-unwinding mechanism of the ring helicase of bacteriophage T7. Proc Natl Acad Sci U S A. 101:7264-9.

Stano NM. Patel SS. (2004) T7 lysozyme represses T7 RNA polymerase transcription by destabilizing the open complex during initiation. J Biol Chem. 279:16136-43.

Jeong YJ. Kim DE. Patel SS. (2004) Nucleotide binding induces conformational changes in Escherichia coli transcription termination factor Rho. J Biol Chem. 279:18370-6.

Reviews

Patel SS. Bandwar RP. (2004) Fluorescence methods for studying the kinetics and thermodynamics of transcription initiation. Methods Enzymol. 370:668-86.

Patel. S. S. (2004) DNA Helicases: Hexameric Enzyme Action in Encylopedia of Biological Chemistry. Edited by Paul Modrich.

Patel S.S.. Bandwar. R. P.. and Levin. M. K. (2003) chapter on "Transient State Kinetics and Computational Analysis of Transcription Initiation" in Kinetic Analysis of Macromolecules: A Practical Approach. Editor: Kenneth A. Johnson.

Levin. M. K. and Patel. S. S. (2003) "Helicases as Motor Proteins" in Molecular Motors Editor Manfred Schliwa. pp 179-198. Wiley-VCH Verlag. GmbH. Weinheim. Germany.