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Smita Patel
Structure-function and dynamics of enzyme-catalyzed processes involved in genome replication and transcription.Research InterestsThe 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 HelicasesUp 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 and Regulation of TranscriptionThe 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. Selected PublicationsSorokina M, Koh HR, Patel SS, Ha T. (2009) Fluoresecent Lifetime Trajectories of a Single Fluorophore Reveal Reaction Intermediates During Transcription Initiation. J. Am. Chem. Soc. 131(28):9630-1. Tang, G-Q., Paratkar, S., and Patel, S.S. (2009) Fluorescence mapping of the open complex of yeast mitochondrial RNA polymerase. J. Biol. Chem. 284(9):5514-22. 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. 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. 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 350 452-75. 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. Reviews Levin M. K., Hingorani M.H., Holmes R.M., Patel S.S. and Carson J.H. (2009) Model-based global analysis of heterogeneous experimental data using gfit. Methods Mol. Biol. , Humana Press, Inc. (link to pdf file) Donmez I, Patel SS. (2006) Mechanisms of a ring shaped helicase. Nucleic Acids Res 2006;34(15):4216-24. (link to pdf file) Patel SS, Donmez I. (2006) Mechanisms of helicases. Minireview J. Biol Chem . 281(27):18265-8. Patel, S. S., Bandwar, R. P., and Levin, M. K. (2002) chapter on "Transient State Kinetics and Computational Analysis of Transcription Initiation" in the book on Kinetic Analysis of Macromolecules: A Practical Approach. Editor: Kenneth A. Johnson. (link to PDF file) Levin, M. K. and Patel, S. S. (2002) chapter on "Helicases as Motor Proteins" in the book on Molecular Motors. Editor Manfred Schliwa. Wiley Publication. (link to PDF file) Patel, S. S. and Picha, K. M. (2000) Structure and Mechanism of Hexameric Helicases. Ann. Rev. Biochem. 69: 651-97. (link to pdf file) Patel Laboratory
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