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Fluorescent Methods to Study Transcription Initiation and Transition into Elongation

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Fluorescent Methods for Molecular Motors

Part of the book series: Experientia Supplementum ((EXS,volume 105))

  • 2012 Accesses

Abstract

The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples.

*These authors made equal contribution to this work.

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Abbreviations

2-AP:

2-Aminopurine fluorescence

bp:

Base pair

DNA:

Deoxyribonucleic acid

ds:

Double stranded

EC:

Elongation complex

FRET:

Förster resonance energy transfer

IC:

Initiation complex

K d :

Equilibrium dissociation constant

mt:

Mitochondrial

NTP:

Nucleoside triphosphate

RNA:

Ribonucleic acid

RNAP:

RNA polymerase

ss:

Single stranded

TAMRA:

Tetramethylrhodamine

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Acknowledgments

This work was supported by the NIH grants GM55310 and GM51966 to S.S.P.

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    Authors and Affiliations

    1. Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA

      Aishwarya P. Deshpande, Shemaila Sultana & Smita S. Patel

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    1. Aishwarya P. Deshpande
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    3. Smita S. Patel
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    Correspondence to Smita S. Patel .

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    Editors and Affiliations

    1. Chromosome Organisation and Dynamics, Max-Planck Institute of Biochemistry, Martinsried, Germany

      Christopher P. Toseland

    2. Department of Cellular Physiology, Ludwig-Maximilians-Universität München, Munich, Germany

      Natalia Fili

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    Deshpande, A.P., Sultana, S., Patel, S.S. (2014). Fluorescent Methods to Study Transcription Initiation and Transition into Elongation. In: Toseland, C., Fili, N. (eds) Fluorescent Methods for Molecular Motors. Experientia Supplementum, vol 105. Springer, Basel. https://doi.org/10.1007/978-3-0348-0856-9_6

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