The protein-DNA contacts in RutR•carAB operator complexes.
Pyrimidine-specific regulation of the upstream carP1 promoter of the carbamoylphosphate synthase operon of Escherichia coli requires numerous trans-acting factors: the allosteric transcription regulator RutR, the nucleoid-associated protein integration host factor, and the trigger enzymes aminopeptidase A and PyrH (UMP-kinase). RutR, a TetR family member, binds far upstream of carP1. Here, we establish a high-resolution contact map of RutR•carP1 complexes for backbone and base-specific contacts, analyze DNA bending, determine the DNA sequence specificity of RutR binding by saturation mutagenesis, demonstrate that uracil but not thymine is the physiologically relevant ligand that inhibits the DNA binding capacity of RutR and build a model of the RutR·operator DNA complex based on the crystal structures of RutR and of the DNA-bound family member QacR. Finally, we test the validity of this model with site-directed mutagenesis of the helix-turn-helix DNA binding motif and in vitro binding studies with the cognate purified mutant RutR proteins.
Nguyen
Phu Le Minh
Ple M
Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel (VUB) and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussel, Belgium.
Bervoets
Indra
I
Maes
Dominique
D
Charlier
Daniel
D
D019943:Amino Acid Substitution; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D017434:Protein Structure, Tertiary; D013941:Thymine / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014498:Uracil / Q000378:metabolism
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; C530619:RutR protein, E coli; D014157:Transcription Factors; D014498:Uracil; D019731:Carbon-Nitrogen Ligases; D013941:Thymine
Proc Natl Acad Sci U S A. 1984 Jul;81(13):4139-43
6377309
Nucleic Acids Res. 2008 Mar;36(5):1567-77
18208836
J Mol Biol. 2005 Sep 16;352(2):438-54
16095620
Nucleic Acids Res. 1988 Oct 25;16(20):9687-705
2972993
Mol Microbiol. 2007 Nov;66(3):744-57
17919280
Proc Natl Acad Sci U S A. 2006 Mar 28;103(13):5114-9
16540542
Nature. 1984 Apr 5-11;308(5959):509-13
6323997
J Mol Biol. 1992 Jul 20;226(2):367-86
1640456
EMBO J. 2002 Mar 1;21(5):1210-8
11867549
J Bacteriol. 2006 May;188(9):3236-45
16621816
Proc Natl Acad Sci U S A. 1985 Mar;82(5):1376-80
2983343
Proc Natl Acad Sci U S A. 1986 Aug;83(15):5469-73
3090544
J Mol Biol. 2000 Sep 15;302(2):411-26
10970742
Nat Struct Biol. 2000 Mar;7(3):215-9
10700280
Nucleic Acids Res. 2007;35(2):623-33
17178749
J Mol Biol. 1998 Apr 10;277(4):805-24
9545374
Nucleic Acids Res. 1988 Aug 11;16(15):7351-67
3045756
J Bacteriol. 1998 Feb;180(3):705-13
9457878
Biochemistry. 1987 Nov 17;26(23):7234-8
3322397
Nucleic Acids Res. 2009 Apr;37(5):1463-76
19136463
Gene. 1989 Dec 21;85(1):15-23
2533576
J Comput Chem. 2004 Oct;25(13):1605-12
15264254
Proc Natl Acad Sci U S A. 1980 Jan;77(1):122-6
6987648
Proc Natl Acad Sci U S A. 1984 Jul;81(13):4134-8
6330744
Mol Microbiol. 2004 Oct;54(2):321-36
15469506
Microbiol Mol Biol Rev. 2005 Jun;69(2):326-56
15944459
Methods Enzymol. 1980;65(1):499-560
6246368
J Mol Biol. 1995 Jul 21;250(4):392-406
7616564
J Mol Biol. 2004 Feb 6;336(1):25-42
14741201
Mol Gen Genet. 1993 Feb;237(1-2):273-86
8455562
Nucleic Acids Res. 2008 Jul;36(12):3950-5
18515344
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
J Mol Biol. 1988 Dec 20;204(4):867-77
3065519
Mol Plant Microbe Interact. 2004 May;17(5):521-31
15141956
J Mol Biol. 1998 Jul 24;280(4):571-82
9677289
Proc Natl Acad Sci U S A. 1987 Oct;84(19):6673-6
2958845