The goal of the proposed studies is to understand the fundamental mechanisms by which single-subunit RNA polymerases (RNAP) catalyze transcription. Single-subunit RNAPs transcribe the genomes of mitochondria that are found in eucaryotes and parasites and mitochondrial RNAPs are remarkably homologous to the RNAPs encoded by T7/T3 like phages. Understanding the mechanisms of single-subunit RNAPs has applications in developing therapeutics for parasitic infectious diseases and diseases related to mitochondrial defects in humans. We propose to investigate the structure and function of single-subunit RNAPs, from bacteriophage T7 and yeast mitochondrial RNAP using approaches such as transient state kinetics and fluorescence resonance energy transfer (FRET). The essential mechanistic features of transcription catalyzed by single subunit or multisubunit RNAPs are conserved. By choosing enzymatically tractable systems, we are able to study the complex mechanisms of transcription in great detail that will eventually lead to a predictive and quantitative model of transcription. It is recognized that transcription initiation, elongation, and termination all play essential roles in regulating gene expression, both in procaryotes and eucaryotes. Dissecting initiation, promoter clearance, and elongation by determining the rate constants and the energetics of the steps provides the essential framework to understand transcription and its regulation. The proposed studies will be carried out with the following specific aims: 1) To characterize the initial steps of transcription initiation in T7 RNAP. We will use a combination of single molecule FRET, stopped- flow FRET, and a rapid protein mapping method to characterize the structures of intermediates and the dynamics of the transition from initiation to elongation. 2) To investigate the mechanism of transcription by S. cerevisiae mitochondrial RNAP. Using established biophysical approaches from our studies of T7 RNAP, we will investigate the initiation mechanisms of mitochondrial RNAP with the goal of understanding the role of its specificity factor. 3) To characterize the kinetic pathway of transcription elongation. Radiometric quenched-flow, fluorescence stopped-flow, and computational kinetic modeling methods will be used to define the elementary steps of nucleotide addition and to understand the mechanisms of fidelity and strand displacement RNA synthesis during elongation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM051966-15
Application #
7663953
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Ikeda, Richard A
Project Start
1995-01-01
Project End
2012-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
15
Fiscal Year
2009
Total Cost
$417,789
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Biochemistry
Type
Schools of Medicine
DUNS #
617022384
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Ramachandran, Aparna; Nandakumar, Divya; Deshpande, Aishwarya P et al. (2016) The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA. J Biol Chem 291:16828-39
Nandakumar, Divya; Patel, Smita S (2015) Finding the right match fast. Cell 160:809-811
Pandey, Manjula; Patel, Smita S (2014) Helicase and polymerase move together close to the fork junction and copy DNA in one-nucleotide steps. Cell Rep 6:1129-1138
Deshpande, Aishwarya P; Patel, Smita S (2014) Interactions of the yeast mitochondrial RNA polymerase with the +1 and +2 promoter bases dictate transcription initiation efficiency. Nucleic Acids Res 42:11721-32
Tang, Guo-Qing; Nandakumar, Divya; Bandwar, Rajiv P et al. (2014) Relaxed rotational and scrunching changes in P266L mutant of T7 RNA polymerase reduce short abortive RNAs while delaying transition into elongation. PLoS One 9:e91859
Deshpande, Aishwarya P; Sultana, Shemaila; Patel, Smita S (2014) Fluorescent methods to study transcription initiation and transition into elongation. Exp Suppl 105:105-30
Ramanagoudr-Bhojappa, Ramanagouda; Chib, Shubeena; Byrd, Alicia K et al. (2013) Yeast Pif1 helicase exhibits a one-base-pair stepping mechanism for unwinding duplex DNA. J Biol Chem 288:16185-95
Hsieh, Fu-Kai; Kulaeva, Olga I; Patel, Smita S et al. (2013) Histone chaperone FACT action during transcription through chromatin by RNA polymerase II. Proc Natl Acad Sci U S A 110:7654-9
Deshpande, Aishwarya P; Patel, Smita S (2012) Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase. Biochim Biophys Acta 1819:930-8
Kim, Hajin; Tang, Guo-Qing; Patel, Smita S et al. (2012) Opening-closing dynamics of the mitochondrial transcription pre-initiation complex. Nucleic Acids Res 40:371-80

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