This proposal seeks to unravel unresolved questions in the mechanisms and regulation of prokaryotic transcription initiation, elongation and termination, as well as in early stages of eukaryotic transcription initiation. Single molecule fluorescence spectroscopic and microscopic methods will be used to detail the sequence, magnitude, kinetics and order of conformational transitions and intermediates along the basal transcription cycle pathway. Transient protein-DNA and protein-protein interactions (and their induced conformations) of the prokaryotic transcription machinery's subunits and of some of the eukaryotic transcription machinery's subunits will be investigated. The mechanisms of prokaryotic and eukaryotic transcription regulation will also be explored using several transcription factors as model systems. Several single molecule assay formats, implemented on freely diffusing transcription complexes and on immobilized complexes will be utilized to address mechanistic and kinetic questions respectively. The proposed studies are expected to elucidate the role of conformational transitions and biomolecular interactions in transcription and its regulation, and the tools developed for these studies could be generalized for the study of regulatory circuits and other biomolecular machineries. The proposed studies of prokaryotic transcription will provide a basis for understanding antibacterial drugs (through inhibition of conformations) and therefore will provide relevant insight and assays for drugs design and screening. The proposed studies of eukaryotic transcription will lay a foundation for understanding mechanisms of transcription related human diseases and will aid in designing and screening therapeutics agents for these diseases.

Public Health Relevance

The proposed work will unravel the detailed mechanism of the first and most important step in gene expression, i.e. transcription and transcription regulation. It will pave the way for detailed molecular and structural understanding of the action of antibacterial agents. It will also provide answers for several long standing and unresolved questions regarding the early stages of eukaryotic transcription initiation and regulation. The latter will provide detailed molecular understanding of the causes for transcription related diseases and will provide tools for drug design and screening that will prevent, manage and cure these diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Lewis, Catherine D
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University of California Los Angeles
Schools of Medicine
Los Angeles
United States
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Ingargiola, Antonino; Segal, Maya; Gulinatti, Angelo et al. (2018) 48-spot single-molecule FRET setup with periodic acceptor excitation. J Chem Phys 148:123304
Lerner, Eitan; Cordes, Thorben; Ingargiola, Antonino et al. (2018) Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer. Science 359:
Lerner, Eitan; Ingargiola, Antonino; Weiss, Shimon (2018) Characterizing highly dynamic conformational states: The transcription bubble in RNAP-promoter open complex as an example. J Chem Phys 148:123315
Ingargiola, Antonino; Peronio, Pietro; Lerner, Eitan et al. (2017) 16-Ch Time-resolved Single-Molecule Spectroscopy Using Line Excitation. Proc SPIE Int Soc Opt Eng 10071:
Ingargiola, Antonino; Lerner, Eitan; Chung, SangYoon et al. (2017) Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules. PLoS One 12:e0175766
Lerner, Eitan; Ingargiola, Antonino; Lee, Jookyung J et al. (2017) Different types of pausing modes during transcription initiation. Transcription 8:242-253
Ingargiola, Antonino; Lerner, Eitan; Chung, SangYoon et al. (2016) FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET. PLoS One 11:e0160716
Lerner, Eitan; Chung, SangYoon; Allen, Benjamin L et al. (2016) Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A 113:E6562-E6571
Ploetz, Evelyn; Lerner, Eitan; Husada, Florence et al. (2016) Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers. Sci Rep 6:33257
Lerner, Eitan; Ploetz, Evelyn; Hohlbein, Johannes et al. (2016) A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement-Förster-Type Resonance Energy Transfer (PIFE-FRET). J Phys Chem B 120:6401-10

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