RNA polymerase supply and flux through the lac operon in Escherichia coli.
Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase.This article is part of the themed issue 'The new bacteriology'.
School of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK.
Bandar
B
Sendy
School of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK.
David J
DJ
Lee
School of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK s.j.w.busby@bham.ac.uk.
Stephen J W
0000-0003-2148-1758
SJ
Busby
School of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK.
Jack A
JA
Bryant
D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D012321:DNA-Directed RNA Polymerases
RNA polymerase; chromatin immunoprecipitation; polymerase density; polymerase per second; promoter occupation; rifampicin
FASEB J. 2006 Aug;20(10):1721-3
16818468
Mol Syst Biol. 2015 Jan 12;11(1):781
25583150
Annu Rev Microbiol. 2012;66:125-52
22726217
Mol Microbiol. 1992 Sep;6(17):2419-22
1328815
Gene Regul Syst Bio. 2007;1:275-93
19119427
Science. 1997 Sep 5;277(5331):1453-62
9278503
Mol Microbiol. 2007 Jul;65(1):21-6
17581117
J Mol Biol. 1983 Jun 25;167(2):259-74
6408264
Methods Enzymol. 1991;208:236-58
1779836
Cell Mol Life Sci. 2007 Jan;64(1):3-16
17103112
Biochim Biophys Acta. 2002 Sep 13;1577(2):191-207
12213652
Nucleic Acids Res. 2000 May 1;28(9):1864-70
10756184
Annu Rev Microbiol. 2000;54:499-518
11018136
Proc Natl Acad Sci U S A. 2015 Aug 11;112(32):E4390-9
26224838
BMC Genomics. 2013 Apr 15;14:254
23586855
Nat Rev Microbiol. 2004 Jan;2(1):57-65
15035009
Cell. 2014 Jan 16;156(1-2):183-94
24361104
Biochem Soc Symp. 2006;(73):1-10
16626282
J Biol Eng. 2009 Mar 20;3:4
19298678
Proc Natl Acad Sci U S A. 1979 Jul;76(7):3194-7
226959
EcoSal Plus. 2008 Sep;3(1):null
26443745
J Bacteriol. 1994 May;176(10 ):2807-13
7514589
J Bacteriol. 2005 Sep;187(17):6166-74
16109958
Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17693-8
16301522
Biochemistry. 2015 Dec 22;54(50):7393-408
26610896
Science. 2014 Jun 13;344(6189):1285-9
24926020
J Biol Chem. 1978 Dec 25;253(24):8949-56
363713
BMC Microbiol. 2009 Dec 09;9:252
20003185
Nat Rev Microbiol. 2016 Oct;14 (10 ):638-50
27498839
Science. 2014 May 30;344(6187):1042-7
24789973
Cell. 2001 Mar 23;104(6):901-12
11290327
Mol Cell. 2011 Feb 4;41(3):249-62
21292158
J Bacteriol. 1991 Jan;173(1):28-36
1702782
Nat Methods. 2015 Aug;12(8):739-42
26098021
Gene. 1991 Jan 2;97(1):7-12
1847348
Science. 2006 Nov 17;314(5802):1144-7
17110578
Methods Enzymol. 2011;498:449-71
21601690
J Mol Biol. 2011 Oct 7;412(5):782-92
21316374
Science. 2006 Nov 17;314(5802):1139-43
17110577
Nat Biotechnol. 2009 Nov;27(11):1043-9
19881496
Science. 2016 Mar 4;351(6277):1090-3
26941320
J Mol Biol. 1984 Dec 25;180(4):881-909
6098691
Proc Natl Acad Sci U S A. 2015 Aug 11;112(32):E4374-80
26216999
Biochim Biophys Acta. 1968 Mar 18;157(1):218-9
4868249
Science. 2016 Apr 1;352(6281):aac7341
27034378
Nucleic Acids Res. 2014 Oct;42(18):11383-92
25209233
Mol Cell. 2006 Dec 8;24(5):747-57
17157257
Mol Microbiol. 2005 Jul;57(1):9-16
15948945