title: Applied force provides insight into transcriptional pausing and its modulation by transcription factor NusA.
abstract: Transcriptional pausing by RNA polymerase (RNAP) plays an essential role in gene regulation. Pausing is modified by various elongation factors, including prokaryotic NusA, but the mechanisms underlying pausing and NusA function remain unclear. Alternative models for pausing invoke blockade events that precede translocation (on-pathway), enzyme backtracking (off-pathway), or isomerization to a nonbacktracked, elemental pause state (off-pathway). We employed an optical trapping assay to probe the motions of individual RNAP molecules transcribing a DNA template carrying tandem repeats encoding the his pause, subjecting these enzymes to controlled forces. NusA significantly decreased the pause-free elongation rate of RNAP while increasing the probability of entry into short- and long-lifetime pauses, in a manner equivalent to exerting a ~19 pN force opposing transcription. The effects of force and NusA on pause probabilities and lifetimes support a reaction scheme where nonbacktracked, elemental pauses branch off the elongation pathway from the pretranslocated state of RNAP.
authors.affiliation: Department of Applied Physics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhou
authors.forename: Kook Sun
authors.initials: KS
authors.lastname: Ha
authors.forename: Arthur
authors.initials: A
authors.lastname: La Porta
authors.forename: Robert
authors.initials: R
authors.lastname: Landick
authors.forename: Steven M
authors.initials: SM
authors.lastname: Block
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D055620:Optical Phenomena; D052898:Optical Tweezers; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D020080:Tandem Repeat Sequences; D013698:Templates, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D012321:DNA-Directed RNA Polymerases
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):8797-8
references.pmid: 19470457
references.citation: Mol Microbiol. 1999 Jan;31(2):651-63
references.pmid: 10027981
references.citation: Cell. 2005 Jan 28;120(2):183-93
references.pmid: 15680325
references.citation: Anal Biochem. 1990 Sep;189(2):254-61
references.pmid: 1704199
references.citation: Science. 1998 Oct 30;282(5390):902-7
references.pmid: 9794753
references.citation: J Biol Chem. 1989 Dec 5;264(34):20796-804
references.pmid: 2479649
references.citation: Phys Rev Lett. 2007 Feb 9;98(6):068103
references.pmid: 17358986
references.citation: Cell. 2002 Apr 19;109(2):193-203
references.pmid: 12007406
references.citation: Genes Dev. 2000 Oct 15;14(20):2664-75
references.pmid: 11040219
references.citation: J Mol Biol. 2004 Nov 19;344(2):335-49
references.pmid: 15522289
references.citation: J Biol Chem. 1981 Mar 25;256(6):2777-86
references.pmid: 7009597
references.citation: J Biol Chem. 1997 Jun 13;272(24):15329-38
references.pmid: 9182561
references.citation: Biochem Soc Trans. 2006 Dec;34(Pt 6):1062-6
references.pmid: 17073751
references.citation: EMBO Rep. 2009 Sep;10(9):997-1002
references.pmid: 19680289
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13538-43
references.pmid: 12370445
references.citation: Science. 1998 Jul 31;281(5377):660-5
references.pmid: 9685251
references.citation: Biophys J. 2005 Dec;89(6):L61-3
references.pmid: 16239336
references.citation: Virology. 1974 Mar;58(1):141-8
references.pmid: 4595374
references.citation: J Mol Biol. 1998 May 1;278(2):307-16
references.pmid: 9571053
references.citation: Cell. 2006 Jun 16;125(6):1083-94
references.pmid: 16777599
references.citation: Nature. 1981 Jul 16;292(5820):215-20
references.pmid: 6265785
references.citation: J Mol Biol. 2008 Oct 10;382(3):628-37
references.pmid: 18647607
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7090-5
references.pmid: 10860976
references.citation: Mol Cell. 2003 Nov;12(5):1125-36
references.pmid: 14636572
references.citation: Mol Microbiol. 1999 Nov;34(3):523-37
references.pmid: 10564494
references.citation: Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1755-60
references.pmid: 9050851
references.citation: Proc Natl Acad Sci U S A. 1985 Jul;82(14):4663-7
references.pmid: 2991886
references.citation: Nucleic Acids Res. 1981 Feb 11;9(3):563-77
references.pmid: 7012794
references.citation: Cell. 2003 Nov 14;115(4):437-47
references.pmid: 14622598
references.citation: Mol Cell. 2001 Jun;7(6):1177-89
references.pmid: 11430821
references.citation: J Biol Chem. 2007 Jun 29;282(26):19020-8
references.pmid: 17502377
references.citation: Biochemistry. 1988 Jul 26;27(15):5622-7
references.pmid: 2846044
references.citation: J Mol Biol. 2010 May 28;399(1):17-30
references.pmid: 20381500
references.citation: Mol Cell. 2011 Feb 4;41(3):249-62
references.pmid: 21292158
references.citation: Annu Rev Biochem. 1997;66:117-72
references.pmid: 9242904
references.citation: Annu Rev Biochem. 2008;77:149-76
references.pmid: 18410247
references.citation: J Biol Chem. 1997 May 9;272(19):12265-71
references.pmid: 9139668
references.citation: Mol Cell. 2006 Jul 21;23(2):231-9
references.pmid: 16857589
references.citation: J Mol Biol. 2001 Aug 10;311(2):265-82
references.pmid: 11478860
references.citation: Nature. 2005 Nov 24;438(7067):460-5
references.pmid: 16284617
references.citation: Science. 2003 Jun 27;300(5628):2061-5
references.pmid: 12791999
references.citation: Mol Cell. 2007 Aug 3;27(3):406-19
references.pmid: 17679091
references.citation: Annu Rev Biophys Biomol Struct. 2006;35:161-75
references.pmid: 16689632
references.citation: Biophys J. 2009 Mar 18;96(6):2189-93
references.pmid: 19289045
references.citation: J Mol Biol. 2010 Sep 3;401(5):708-25
references.pmid: 20600118
references.citation: Bioessays. 2002 Aug;24(8):700-7
references.pmid: 12210530
references.citation: J Mol Biol. 2006 Mar 10;356(5):1163-79
references.pmid: 16405998
references.citation: Annu Rev Microbiol. 2008;62:211-33
references.pmid: 18729732
references.citation: Mol Cell. 2009 Jan 16;33(1):97-108
references.pmid: 19150431
references.citation: Nature. 2007 Apr 12;446(7137):820-3
references.pmid: 17361130
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):8900-5
references.pmid: 19416863
references.citation: Nature. 2003 Dec 11;426(6967):684-7
references.pmid: 14634670
references.citation: Science. 2001 Apr 27;292(5517):730-3
references.pmid: 11326100
references.citation: J Mol Biol. 1987 Jun 20;195(4):809-18
references.pmid: 2821282