title: Additional determinants within Escherichia coli FNR activating region 1 and RNA polymerase alpha subunit required for transcription activation.
abstract: The global anaerobic regulator FNR is a DNA binding protein that activates transcription of genes required for anaerobic metabolism in Escherichia coli through interactions with RNA polymerase (RNAP). Alanine-scanning mutagenesis of FNR amino acid residues 181 to 193 of FNR was utilized to determine which amino acid side chains are required for transcription of both class II and class I promoters. In vivo assays of FNR function demonstrated that a core of residues (F181, R184, S187, and R189) was required for efficient activation of class II promoters, while at a class I promoter, FF(-61.5), only S187 and R189 were critical for FNR activation. Site-directed mutagenesis of positions 184, 187, and 189 revealed that the positive charge contributes to the function of the side chain at positions 184 and 189 while the serine hydroxyl is critical for the function of position 187. Subsequent analysis of the carboxy-terminal domain of the alpha subunit (alphaCTD) of RNAP, using an alanine library in single copy, revealed that in addition to previously characterized side chains (D305, R317, and L318), E286 and E288 contributed to FNR activation of both class II and class I promoters, suggesting that alphaCTD region 285 to 288 also participates in activation by FNR. In conclusion, this study demonstrates that multiple side chains within region 181 to 192 are required for FNR activation and the surface of alphaCTD required for FNR activation is more extensive than previously observed.
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, USA.
authors.forename: K Derek
authors.initials: KD
authors.lastname: Weber
authors.forename: Owen D
authors.initials: OD
authors.lastname: Vincent
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Kiley
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit; D000409:Alanine
references.citation: J Mol Biol. 1995 Jan 27;245(4):351-61
references.pmid: 7837268
references.citation: J Mol Biol. 1994 Nov 4;243(4):603-10
references.pmid: 7966285
references.citation: J Biol Chem. 1996 Feb 2;271(5):2762-8
references.pmid: 8576252
references.citation: Mol Microbiol. 1996 Jan;19(1):125-37
references.pmid: 8821942
references.citation: Nucleic Acids Res. 1997 Jan 15;25(2):326-32
references.pmid: 9016561
references.citation: Nucleic Acids Res. 1997 Oct 15;25(20):4028-34
references.pmid: 9321653
references.citation: Nucleic Acids Res. 1998 May 1;26(9):2075-81
references.pmid: 9547262
references.citation: EMBO J. 1998 Jun 15;17(12):3439-47
references.pmid: 9628879
references.citation: J Mol Biol. 1998 Dec 18;284(5):1353-65
references.pmid: 9878355
references.citation: J Biol Chem. 1999 Apr 16;274(16):10840-5
references.pmid: 10196160
references.citation: J Biol Chem. 1999 Apr 9;274(15):10244-8
references.pmid: 10187810
references.citation: J Bacteriol. 2005 Feb;187(3):1135-60
references.pmid: 15659690
references.citation: Mol Microbiol. 2000 Sep;37(5):1032-40
references.pmid: 10972822
references.citation: J Biol Chem. 2000 Sep 15;275(37):29061-5
references.pmid: 10846171
references.citation: Mol Microbiol. 2000 Nov;38(4):817-27
references.pmid: 11115116
references.citation: Essays Biochem. 2001;37:17-31
references.pmid: 11758454
references.citation: J Mol Biol. 2002 Jan 18;315(3):275-83
references.pmid: 11786011
references.citation: J Mol Biol. 2002 Feb 22;316(3):501-16
references.pmid: 11866514
references.citation: J Bacteriol. 2002 Apr;184(8):2273-80
references.pmid: 11914359
references.citation: Science. 2002 Aug 30;297(5586):1562-6
references.pmid: 12202833
references.citation: J Bacteriol. 2002 Nov;184(21):5855-61
references.pmid: 12374818
references.citation: FEMS Microbiol Rev. 2003 Dec;27(5):559-92
references.pmid: 14638413
references.citation: J Biol Chem. 2003 Dec 26;278(52):52944-52
references.pmid: 14530288
references.citation: Methods Enzymol. 2003;370:300-12
references.pmid: 14712655
references.citation: FEBS Lett. 2004 Jan 30;558(1-3):13-8
references.pmid: 14759508
references.citation: J Bacteriol. 1978 Jun;134(3):1141-56
references.pmid: 149110
references.citation: J Mol Biol. 1987 Nov 20;198(2):311-26
references.pmid: 2828639
references.citation: Genes Dev. 1989 Sep;3(9):1462-71
references.pmid: 2691330
references.citation: J Bacteriol. 1991 Dec;173(23):7511-8
references.pmid: 1938946
references.citation: Nucleic Acids Res. 1991 Dec 25;19(24):6705-12
references.pmid: 1762901
references.citation: Genes Dev. 1993 Oct;7(10):1993-2005
references.pmid: 8406003
references.citation: Mol Microbiol. 1994 Jan;11(2):383-90
references.pmid: 8170400
references.citation: J Mol Biol. 1994 Nov 4;243(4):595-602
references.pmid: 7966284
references.citation: J Bacteriol. 1995 Dec;177(23):6704-10
references.pmid: 7592457