In Vitro and In Vivo Methodologies for Studying the Sigma 54-Dependent Transcription
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 wordsTranscription activation RNA polymerase σ54 Open and closed promoter complexes AAA+ proteins Bacterial enhancer binding proteins ATPase
This work was supported by BBSRC (BB/J002828/1), Wellcome Trust (WT093044MA), and Leverhulme Trust (F/07 058/BM) project grants.
- 23.Sarkar G, Edery I, Sonenberg N (1985) Photoaffinity labeling of the cap-binding protein complex with ATP/dATP. Differential labeling of free eukaryotic initiation factor 4A and the eukaryotic initiation factor 4A component of the cap-binding protein complex with [alpha-32P]ATP/dATP. J Biol Chem 260:13831–13837PubMedGoogle Scholar
- 27.Miller JH (1992) A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar