ExsA recruits RNA polymerase to an extended -10 promoter by contacting region 4.2 of sigma-70.
ExsA is a member of the AraC family of transcriptional activators and is required for expression of the Pseudomonas aeruginosa type III secretion system (T3SS). ExsA-dependent promoters consist of two binding sites for monomeric ExsA located approximately 50 bp upstream of the transcription start sites. Binding to both sites is required for recruitment of sigma(70)-RNA polymerase (RNAP) to the promoter. ExsA-dependent promoters also contain putative -35 hexamers that closely match the sigma(70) consensus but are atypically spaced 21 or 22 bp from the -10 hexamer. Because several nucleotides located within the putative -35 region are required for ExsA binding, it is unclear whether the putative -35 region makes an additional contribution to transcription initiation. In the present study we demonstrate that the putative -35 hexamer is dispensable for ExsA-independent transcription from the P(exsC) promoter and that deletion of sigma(70) region 4.2, which contacts the -35 hexamer, has no effect on ExsA-independent transcription from P(exsC). Region 4.2 of sigma(70), however, is required for ExsA-dependent activation of the P(exsC) and P(exsD) promoters. Genetic data suggest that ExsA directly contacts region 4.2 of sigma(70), and several amino acids were found to contribute to the interaction. In vitro transcription assays demonstrate that an extended -10 element located in the P(exsC) promoter is important for overall promoter activity. Our collective data suggest a model in which ExsA compensates for the lack of a -35 hexamer by interacting with region 4.2 of sigma(70) to recruit RNAP to the promoter.
Vakulskas
Christopher A
CA
Department of Microbiology, University of Iowa, Iowa City, IA, USA.
Brutinel
Evan D
ED
Yahr
Timothy L
TL
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C095093:ExsA protein, Pseudomonas aeruginosa; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D015534:Trans-Activators; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases
Cell. 1994 Sep 9;78(5):889-96
8087855
J Biol Chem. 2004 Apr 23;279(17):17397-403
14976210
Mol Microbiol. 2003 Dec;50(5):1683-701
14651647
Mol Microbiol. 2002 Mar;43(6):1543-53
11971264
Infect Control Hosp Epidemiol. 2000 Aug;21(8):510-5
10968716
J Biol Chem. 1985 Mar 25;260(6):3539-41
2579077
J Bacteriol. 2000 Jun;182(12):3529-35
10852886
Proc Natl Acad Sci U S A. 2000 Nov 21;97(24):13215-20
11087868
Biotechniques. 2000 Nov;29(5):948-50, 952
11084852
Infect Immun. 1995 Apr;63(4):1541-51
7890421
Appl Environ Microbiol. 2009 May;75(9):2705-11
19251886
Mol Microbiol. 2006 Nov;62(3):631-40
16995895
J Bacteriol. 2007 Nov;189(22):8387-91
17827287
J Bacteriol. 2009 Jun;191(12):3811-21
19376850
Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):472-6
8380640
Nat Rev Microbiol. 2004 Jan;2(1):57-65
15035009
Methods Enzymol. 1996;273:130-4
8791605
Nucleic Acids Res. 1997 Jan 15;25(2):326-32
9016561
J Bacteriol. 2008 Mar;190(6):2249-51
18192384
J Bacteriol. 2004 Sep;186(18):6277-85
15342598
J Mol Biol. 1996 Mar 1;256(3):436-48
8604129
J Bacteriol. 1995 Dec;177(24):7100-4
8522515
J Bacteriol. 1999 Aug;181(16):4949-54
10438766
Gene. 1981 Jun-Jul;14(1-2):63-72
7021324
J Bacteriol. 1999 Sep;181(17):5185-92
10464186
J Bacteriol. 1991 Mar;173(6):1944-50
2001998
J Bacteriol. 2009 Nov;191(21):6654-64
19717612
Mol Microbiol. 2002 Nov;46(4):1123-33
12421316
EMBO J. 2001 Apr 17;20(8):1850-62
11296219
J Bacteriol. 2010 Mar;192(6):1479-86
20008065
J Bacteriol. 2000 Sep;182(17):4959-69
10940041
Mol Cell. 2002 Mar;9(3):527-39
11931761
Nucleic Acids Res. 2003 Aug 15;31(16):4689-95
12907708
EMBO J. 1998 Jun 15;17(12):3439-47
9628879
J Bacteriol. 1999 Aug;181(15):4704-7
10419977
Curr Opin Microbiol. 2001 Apr;4(2):132-7
11282467
J Bacteriol. 1995 Mar;177(5):1169-78
7868588
Mol Microbiol. 2008 May;68(3):657-71
18373522
Mol Microbiol. 1997 Nov;26(4):621-9
9427393
J Bacteriol. 2004 Feb;186(3):631-7
14729687
J Biol Chem. 2009 Jun 5;284(23):15762-70
19369699
J Mol Biol. 1983 Jun 5;166(4):557-80
6345791
J Biol Chem. 1956 Jan;218(1):97-106
13278318
Mol Microbiol. 2003 May;48(4):863-74
12753182
Mol Microbiol. 2004 Sep;53(5):1279-90
15387809
J Bacteriol. 2000 Dec;182(24):7075-7
11092872
Mol Microbiol. 2004 Oct;54(2):287-90
15469502
EMBO J. 1997 Jul 1;16(13):4034-40
9233812
Mol Microbiol. 2004 Mar;51(5):1297-309
14982625
Infect Immun. 2007 Sep;75(9):4432-9
17635873
J Bacteriol. 2001 Nov;183(21):6384-93
11591683
Mol Microbiol. 2004 Jul;53(1):297-308
15225323
Plasmid. 2000 Jan;43(1):59-72
10610820
J Bacteriol. 2008 May;190(9):3110-7
18296515
Infect Immun. 1994 Feb;62(2):554-63
8300213
Mol Microbiol. 2008 Aug;69(3):747-64
18554322
Gene. 1999 Feb 18;227(2):197-203
10023058
J Bacteriol. 1994 Jul;176(13):3832-38
8021164
J Bacteriol. 1995 Aug;177(15):4427-36
7635828
J Mol Biol. 1993 Jul 20;232(2):406-18
8345519
Crit Care Med. 1999 May;27(5):887-92
10362409
J Bacteriol. 1991 Oct;173(20):6460-8
1655713
Rev Physiol Biochem Pharmacol. 2004;152:79-92
15375697
EMBO J. 1994 Oct 3;13(19):4558-67
7925297
Burns. 2001 Mar;27(2):129-30
11226648
J Mol Biol. 1998 Dec 18;284(5):1353-65
9878355
Mol Microbiol. 1993 May;8(5):797-802
8394979
FEMS Microbiol Lett. 1999 Dec 15;181(2):307-12
10585554
Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13899-904
9811898
Mol Microbiol. 2005 Jun;56(6):1627-35
15916611
J Mol Biol. 1982 May 25;157(3):493-525
6214638