The goal of this research project is to understand the fundamental mechanisms by which single-subunit RNA polymerases catalyze transcription. Single-subunit RNA polymerases are widespread in nature transcribing not only the genomes of phages but also the genomes of mitochondria that are vital for energy production and survival of eukaryotes including humans. Understanding the molecular mechanisms of gene expression by single-subunit RNA polymerases has applications in developing therapeutics for numerous mitochondrial related human diseases as well as combating parasitic infections. It is recognized that transcription initiation, elongation, and termination all play essential roles in regulating gene expression. However, we lack quantitative description of these conserved transcriptional processes. By choosing enzymatically tractable systems, such as T7 RNA polymerase and mitochondrial RNA polymerase of the yeast, we are able to obtain in depth knowledge of each stage to ultimately develop a predictive and quantitative model of transcription. There is a need to develop new methods to assay the various steps of transcription initiation and elongation in real time that identify key intermediates and measure the rate constants and energetics of the elementary steps. One of our goals is to develop fluorescence-based transient state kinetic approaches that can be used in ensemble and single molecule experimental set up to measure the dynamics of transcription in real time. These methods will be developed through studies of T7 RNA polymerase and applied to understand the mechanism of mitochondrial RNA polymerases and associated factors. Unlike T7 RNA polymerase, the mitochondrial RNA polymerases require transcription factors, and one of our goals is to understand how these accessory factors aid in transcription and its regulation. The proposed studies will determine 1) the mechanism of polymerase translocation and fidelity of transcription elongation, 2) role ofthe transcription factors and accessory factors in various steps of transcription by the mitochondrial RNA polymerases, and 3) role of transcription in the initiation of replication.

Public Health Relevance

Single-subunit RNA polymerases transcribe the genomes of mitochondria including humans. This proposal addresses the basic mechanisms of transcription by single-subunit RNA polymerases, which will provide the knowledge to develop new ways to diagnose and treat human mitochondrial diseases such as Parkinson's, Alzheimer's, and type 2 diabetes, and to combat parasitic infectious diseases such as Malaria.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Barski, Oleg
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University of Medicine & Dentistry of NJ
Schools of Medicine
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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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