title: Insights into the mechanism of initial transcription in Escherichia coli RNA polymerase.
abstract: It has long been known that during initial transcription of the first 8-10 bases of RNA, complexes are relatively unstable, leading to the release of short abortive RNA transcripts. An early "stressed intermediate" model led to a more specific mechanistic model proposing "scrunching" stress as the basis for the instability. Recent studies in the single subunit T7 RNA polymerase have argued against scrunching as the energetic driving force and instead argue for a model in which pushing of the RNA-DNA hybrid against a protein element associated with promoter binding, while likely driving promoter release, reciprocally leads to instability of the hybrid. In this study, we test these models in the structurally unrelated multisubunit bacterial RNA polymerase. Via the targeted introduction of mismatches and nicks in the DNA, we demonstrate that neither downstream bubble collapse nor compaction/scrunching of either the single-stranded template or nontemplate strands is a major force driving abortive instability (although collapse from the downstream end of the bubble does contribute significantly to the instability of artificially halted complexes). In contrast, pushing of the hybrid against a mobile protein element (σ3.2 in the bacterial enzyme) results in substantially increased abortive instability and is likely the primary energetic contributor to abortive cycling. The results suggest that abortive instability is a by-product of the mechanistic need to couple the energy of nucleotide addition (RNA chain growth) to driving the timed release of promoter contacts during initial transcription.
authors.affiliation: From the Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003.
authors.forename: Satamita
authors.initials: S
authors.lastname: Samanta
authors.forename: Craig T
authors.initials: CT
authors.lastname: Martin
mesh_terms: D053904:DNA Breaks, Single-Stranded; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D009692:Nucleic Acid Heteroduplexes / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D012329:RNA, Bacterial; D012321:DNA-Directed RNA Polymerases
keywords: DNA-Protein Interaction; Enzyme Mechanisms; Enzymology; Nucleic Acid Enzymology; RNA; RNA Polymerase; Transcription
references.citation: Science. 2004 Feb 13;303(5660):1014-6
references.pmid: 14963331
references.citation: J Mol Biol. 1973 Jun 25;77(2):255-77
references.pmid: 4587752
references.citation: Biochemistry. 1996 Nov 19;35(46):14574-82
references.pmid: 8931555
references.citation: J Biol Chem. 2006 Jul 7;281(27):18273-6
references.pmid: 16690607
references.citation: Biochemistry. 1993 Apr 27;32(16):4281-5
references.pmid: 8476857
references.citation: Science. 2011 Jul 29;333(6042):633-7
references.pmid: 21798951
references.citation: Biochemistry. 2003 Apr 8;42(13):3777-86
references.pmid: 12667069
references.citation: Nature. 1999 May 6;399(6731):80-3
references.pmid: 10331394
references.citation: J Biol Chem. 2012 Oct 26;287(44):37352-61
references.pmid: 22923611
references.citation: J Mol Biol. 1994 Feb 11;236(1):72-80
references.pmid: 7508986
references.citation: J Biol Chem. 1998 Apr 17;273(16):9872-7
references.pmid: 9545328
references.citation: Biochemistry. 1980 Jul 8;19(14):3245-53
references.pmid: 6996702
references.citation: Biochemistry. 2006 Jul 25;45(29):8841-54
references.pmid: 16846227
references.citation: Cell. 2000 Jun 9;101(6):601-11
references.pmid: 10892647
references.citation: Methods. 2009 Jan;47(1):25-36
references.pmid: 18948204
references.citation: J Biol Chem. 1997 Jun 13;272(24):15329-38
references.pmid: 9182561
references.citation: Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):8972-8
references.pmid: 11481468
references.citation: Science. 2008 Oct 24;322(5901):553-7
references.pmid: 18948533
references.citation: J Mol Biol. 2006 Aug 25;361(4):644-58
references.pmid: 16876197
references.citation: Prog Nucleic Acid Res Mol Biol. 2005;80:323-47
references.pmid: 16164978
references.citation: J Bacteriol. 1998 Jun;180(12):3019-25
references.pmid: 9620948
references.citation: EMBO J. 1997 Dec 15;16(24):7468-80
references.pmid: 9405375
references.citation: Biochemistry. 1980 Dec 9;19(25):5864-9
references.pmid: 6450614
references.citation: Biochemistry. 2007 Nov 6;46(44):12687-99
references.pmid: 17929835
references.citation: Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11655-60
references.pmid: 9751721
references.citation: Mol Cell. 2005 Jul 1;19(1):101-10
references.pmid: 15989968
references.citation: Science. 1999 Dec 17;286(5448):2305-9
references.pmid: 10600732
references.citation: J Mol Biol. 2000 Jun 2;299(2):325-35
references.pmid: 10860741
references.citation: Science. 2002 May 17;296(5571):1280-4
references.pmid: 12016306
references.citation: Science. 2012 Nov 23;338(6110):1076-80
references.pmid: 23086998
references.citation: Science. 2002 May 17;296(5571):1285-90
references.pmid: 12016307
references.citation: J Biol Chem. 1998 Sep 4;273(36):23558-66
references.pmid: 9722595
references.citation: EMBO J. 1986 Nov;5(11):2995-3000
references.pmid: 3539590
references.citation: Science. 2006 Nov 17;314(5802):1144-7
references.pmid: 17110578
references.citation: J Biol Chem. 2011 Jun 24;286(25):22600-8
references.pmid: 21525530
references.citation: Biochemistry. 1988 May 31;27(11):3966-74
references.pmid: 3415967
references.citation: Cold Spring Harb Symp Quant Biol. 1998;63:263-7
references.pmid: 10384290
references.citation: Science. 2006 Nov 17;314(5802):1139-43
references.pmid: 17110577
references.citation: J Mol Biol. 2006 Aug 25;361(4):634-43
references.pmid: 16781733
references.citation: Nature. 2013 Jan 17;493(7432):437-40
references.pmid: 23151482
references.citation: Nucleic Acids Res. 1987 Nov 11;15(21):8783-98
references.pmid: 3684574
references.citation: Nature. 2002 Jun 13;417(6890):712-9
references.pmid: 12000971
references.citation: Biochemistry. 2011 Aug 16;50(32):7015-22
references.pmid: 21776950
references.citation: J Biol Chem. 2006 Aug 18;281(33):23533-44
references.pmid: 16790422
references.citation: J Biol Chem. 2003 Feb 21;278(8):5539-47
references.pmid: 12477716
references.citation: Cell. 1997 Apr 4;89(1):33-41
references.pmid: 9094712
references.citation: Cell. 1993 Feb 12;72(3):459-66
references.pmid: 8431948
references.citation: EMBO J. 1986 Nov;5(11):2987-94
references.pmid: 3539589
references.citation: J Biol Chem. 1996 Aug 2;271(31):18775-9
references.pmid: 8702534
references.citation: Cell. 2002 Mar 8;108(5):599-614
references.pmid: 11893332
references.citation: Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11588-92
references.pmid: 8524809
references.citation: J Biol Chem. 2011 Jan 7;286(1):270-9
references.pmid: 20952386
references.citation: J Mol Biol. 2007 Mar 23;367(2):344-57
references.pmid: 17275836
references.citation: J Mol Biol. 2001 Aug 10;311(2):265-82
references.pmid: 11478860
references.citation: J Mol Biol. 1987 Jan 20;193(2):267-78
references.pmid: 2439694
references.citation: Cell. 2003 Aug 8;114(3):335-45
references.pmid: 12914698