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In Vitro and In Vivo Methodologies for Studying the Sigma 54-Dependent Transcription

  • Martin BuckEmail author
  • Christoph Engl
  • Nicolas Joly
  • Goran Jovanovic
  • Milija Jovanovic
  • Edward Lawton
  • Christopher McDonald
  • Jörg Schumacher
  • Christopher Waite
  • Nan Zhang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1276)

Abstract

Here we describe approaches and methods to assaying in vitro the major variant bacterial sigma factor, Sigma 54 (σ54), in a purified system. We include the complete transcription system, binding interactions between σ54 and its activators, as well as the self-assembly and the critical ATPase activity of the cognate activators which serve to remodel the closed promoter complexes. We also present in vivo methodologies that are used to study the impact of physiological processes, metabolic states, global signalling networks, and cellular architecture on the control of σ54-dependent gene expression.

Key words

Transcription activation RNA polymerase σ54 Open and closed promoter complexes AAA+ proteins Bacterial enhancer binding proteins ATPase 

Notes

Acknowledgements

This work was supported by BBSRC (BB/J002828/1), Wellcome Trust (WT093044MA), and Leverhulme Trust (F/07 058/BM) project grants.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Martin Buck
    • 1
    Email author
  • Christoph Engl
    • 1
  • Nicolas Joly
    • 1
    • 2
  • Goran Jovanovic
    • 1
  • Milija Jovanovic
    • 1
  • Edward Lawton
    • 1
  • Christopher McDonald
    • 1
  • Jörg Schumacher
    • 1
  • Christopher Waite
    • 1
  • Nan Zhang
    • 1
  1. 1.Department of Life SciencesImperial College LondonLondonUK
  2. 2.Equipe Nanomanipulation de Biomolecules, Institut Jacques Monod – UMR7592CNRS – Universite ParisParis Cedex 13France

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