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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. 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. T7 RNA polymerase with its 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. 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 PublicationsVanLoock, M. S., Chen, Y., Yu, X., Patel, S. S. and Egelman, E. H. (2001) The primase active site is on the outside of the bacteriophage T7 gene 4 helicase-primase hexameric ring. J. Mol. Biol. 311, 951-956. Stano, N. and Patel, S. S. (2002) The intercalating beta-haipin of T7 RNA polymerase stabilizes the open complex and promotes efficient synthesis of RNA during initiation. J. Mol. Biol. 315, 1009-1025 Bandwar, R., Jia Y., Stano, N. and Patel, S. S. (2002) Kinetic And Thermodynamic Basis Of Promoter Strength: Multiple Steps of Transcription Initiation by T7 RNA Polymerase are Modulated by the Promoter Sequence. Biochemistry, 41, 3586-3595. Kim, D-E and Patel, S. S. (2002) T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA. J. Mol. Biol. 321 807-819. Levin, M. K. and Patel, S. S. (2002) Helicase from Hepatitis C virus: Energetics of DNA binding. J. Biol.Chem. 277: 29377-29385 Stano, N. M. Levin, M. K., and Patel, S. S. (2002) The +2 NTP binding drives open complex formation in T7 RNA polymerase. J. Biol. Chem. 277, 37292-300. Bandwar, R. and Patel, S. S. (2002) Energetics of open complex formation in T7 RNA polymerase. J. Mol. Biol. 324, 63-72. Jeong, Y-J., Kim, D-E., and Patel, S. S. (2002) Kinetic pathway of dTTP hydrolysis by bacteriophate T7 helicase-primase in the absence of DNA. J. Biol. Chem. 277, 43778-43784. 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 (Review). Kim, D-E and Patel, S. S. (2001) The kinetic pathway of RNA binding to the Escherichia coli transcription termination factor Rho. J. Biol. Chem. 13902-13910 Bandwar, R. and Patel, S. S. (2001) Peculiar 2-aminopurine fluorescence monitors the dynamics of open complex formation by bacteriophage T7 RNA polymerase. J. Biol. Chem. 14075-14082. Ahnert, P., Picha, K. M., Patel, S. S. (2000) A ring opening mechanism for DNA binding in the central channel of T7 helicase-primase protein. EMBO J. 19: 3418-3427. Picha, K.M., Ahnert, P. and Patel, S. S. (2000) DNA binding in the central channel of bacteriophage T7 helicase-primase is a multistep process. Nucleotide hydrolysis is not required. Biochemistry, 39: 6401-6409. Levin, M. K. and Patel, S. S. (1999) The helicase from hepatitis C virus is active as an oligomer J. Biol. Chem. 274:31839-31846. Picha M. K. and Patel, S. S. (1998) Bacteriophage T7 DNA helicase binds dTTP, forms hexamers, and binds DNA in the absence of Mg2+: The presence of dTTP is sufficient for hexamer formation and DNA binding. J. Biol. Chem. 273: 27315-27319. Hingorani, M. M., Washington, T. M., Moore, K. C., Patel, S. S. (1997) The dTTPase mechanism of T7 DNA helicase resembles the rotational mechanism of F1-ATPase Proc. Natl. Acad. Sci. 94: 5012-5017. Yiping, J. and Patel, S. S. (1997) Kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase. Biochemistry, 36: 4223-4232. Kumar, A., and Patel, S. S. (1997) Inhibition of T7 RNA polymerase: Transcription initiation and transition from initiation to elongation are inhibited by T7 Lysozyme via a ternary Complex with RNA polymerase and promoter DNA. Biochemistry, 36: 13954-13962. Jia, Y., and Patel, S. S. (1997) Kinetic studies of GTP binding and RNA synthesis during transcription initiation by T7 RNA polymerase. J. Biol. Chem. 272: 30147-30153. Ahnert, P., and Patel, S. S. (1997) Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5'- and 3'-tails of the forked DNA substrate. J. Biol. Chem. 272: 32267-32273. Yu, X., Hingorani, M. M., Patel, S. S., Egelman, E. H. (1996) DNA is bound within the central hole to one or two of the six subunits of the T7 DNA helicase" Nature Struct. Biol. 3: 740-743. Washington, T. M., Rosenberg, A. H., Studier, F. W., and Patel, S. S. (1996) Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis, and the coupling of hydrolysis with DNA unwinding. J. Biol. Chem. 271: 26825-26834. Yiping, J., Kumar, A, and Patel, S. S. (1996) Equilibrium and stopped-flow kinetic studies of interaction between T7 RNA polymerase and its promoters measured by protein and 2-aminopurine fluorescence changes. J. Biol. Chem. 271: 30451-30458. Egelman, E. H., Yu, X., Wild, R., Hingorani, M. M., and Patel, S. S. (1995) T7 helicase/primase proteins form rings around single-stranded DNA that suggests a general structure for hexameric helicases." Proc. Natl. Acad. Sci.U.S.A. 92: 3869-3873. Patel Laboratory
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