Abstract
Bacterial RNA polymerase is the first point of gene expression and a validated target for antibiotics. Studied for several decades, the Escherichia coli transcriptional apparatus is by far the best characterized, with numerous RNA polymerase mutants and auxiliary factors isolated and analyzed in great detail. Since the E. coli enzyme was refractory to crystallization, structural studies have been focused on Thermus RNA polymerases, revealing atomic details of the catalytic center and RNA polymerase interactions with nucleic acids, antibiotics, and regulatory proteins. However, numerous differences between these enzymes, including resistance of Thermus RNA polymerases to some antibiotics, underscored the importance of the E. coli enzyme structures. Three groups published these long awaited structures in 2013, enabling functional and structural studies of the same model system. This progress was made possible, in large part, by the use of multicistronic vectors for expression of the E. coli enzyme in large quantities and in a highly active form. Here we describe the commonly used vectors and procedures for purification of the E. coli RNA polymerase.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Zhang G, Campbell EA, Minakhin L et al (1999) Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 A resolution. Cell 98:811–824
Vassylyev DG, Vassylyeva MN, Zhang J et al (2007) Structural basis for substrate loading in bacterial RNA polymerase. Nature 448:163–168
Zhang Y, Feng Y, Chatterjee S et al (2012) Structural basis of transcription initiation. Science 338:1076–1080
Vassylyev DG, Vassylyeva MN, Perederina A et al (2007) Structural basis for transcription elongation by bacterial RNA polymerase. Nature 448:157–162
Vassylyev DG, Sekine S, Laptenko O et al (2002) Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution. Nature 417:712–719
Tagami S, Sekine S, Kumarevel T et al (2010) Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein. Nature 468:978–982
Belogurov GA, Vassylyeva MN, Sevostyanova A et al (2009) Transcription inactivation through local refolding of the RNA polymerase structure. Nature 457:332–335
Campbell EA, Korzheva N, Mustaev A et al (2001) Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 104:901–912
Vassylyev DG, Svetlov V, Vassylyeva MN et al (2005) Structural basis for transcription inhibition by tagetitoxin. Nat Struct Mol Biol 12:1086–1093
Lane WJ, Darst SA (2010) Molecular evolution of multisubunit RNA polymerases: structural analysis. J Mol Biol 395:686–704
Artsimovitch I, Patlan V, Sekine S et al (2004) Structural basis for transcription regulation by alarmone ppGpp. Cell 117:299–310
Ross W, Vrentas CE, Sanchez-Vazquez P et al (2013) The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation. Mol Cell 50:420–429
Zuo Y, Wang Y, Steitz TA (2013) The mechanism of E. coli RNA polymerase regulation by ppGpp is suggested by the structure of their complex. Mol Cell 50:430–436
Murakami KS (2013) X-ray crystal structure of Escherichia coli RNA polymerase sigma70 holoenzyme. J Biol Chem 288:9126–9134
Bae B, Davis E, Brown D et al (2013) Phage T7 Gp2 inhibition of Escherichia coli RNA polymerase involves misappropriation of sigma70 domain 1.1. Proc Natl Acad Sci U S A 110:19772–19777
Artsimovitch I, Svetlov V, Murakami KS et al (2003) Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions. J Biol Chem 278:12344–12355
Vrentas CE, Gaal T, Ross W et al (2005) Response of RNA polymerase to ppGpp: requirement for the omega subunit and relief of this requirement by DksA. Genes Dev 19:2378–2387
Belogurov GA, Vassylyeva MN, Svetlov V et al (2007) Structural basis for converting a general transcription factor into an operon-specific virulence regulator. Mol Cell 26:117–129
Twist KA, Husnain SI, Franke JD et al (2011) A novel method for the production of in vivo-assembled, recombinant Escherichia coli RNA polymerase lacking the alpha C-terminal domain. Protein Sci 20:986–995
Weilbaecher R, Hebron C, Feng G et al (1994) Termination-altering amino acid substitutions in the beta′ subunit of Escherichia coli RNA polymerase identify regions involved in RNA chain elongation. Genes Dev 8:2913–2927
Yuzenkova J, Delgado M, Nechaev S et al (2002) Mutations of bacterial RNA polymerase leading to resistance to microcin j25. J Biol Chem 277:50867–50875
Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234
Acknowledgements
This work was supported by the National Institutes of Health GM67153 grant.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Svetlov, V., Artsimovitch, I. (2015). Purification of Bacterial RNA Polymerase: Tools and Protocols. In: Artsimovitch, I., Santangelo, T. (eds) Bacterial Transcriptional Control. Methods in Molecular Biology, vol 1276. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2392-2_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2392-2_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2391-5
Online ISBN: 978-1-4939-2392-2
eBook Packages: Springer Protocols