Curation Information

Publication
Genomic analysis of LexA binding reveals the permissive nature of the Escherichia coli genome and identifies unconventional target sites.;Wade JT, Reppas NB, Church GM, Struhl K;Genes & development 2005 Nov 1; 19(21):2619-30 [16264194]
TF
LexA [P0A7C2, view regulon]
Reported TF sp.
Escherichia coli str. K-12 substr. MG1655
Reported site sp.
Escherichia coli str. K-12 substr. MG1655
Created by
Erill Lab
Curation notes
-

Experimental Process

ChIP-chip peak sequence passed to MEME. Motif used with ScanACE to search.

ChIP assay conditions
E. coli strains MG1655 and MG1655 lexA1 were used for ChIP experiments. Cells were grown to mid-exponential phase (OD650 = 0.3-0.6) in LB.
ChIP notes
Cells were grown in appropriate media, and formaldehyde was added to a final concentration of 1%. After 20 min of incubation, glycine was added to a final concentration of 0.5 M, and cells were harvested by centrifugation and washed once with Tris-buffered saline (pH 7.5). Cells were resuspended in 500 μL of lysis buffer (10 mM Tris at pH 8.0, 20% sucrose, 50 mM NaCl, 10 mM EDTA, 4 mg/mL lysozyme) and incubated at 37°C for 30 min. Five-hundred microliters of immunoprecipitation (IP) buffer (50 mM HEPES-KOH at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) and PMSF (final concentration 1 mM) were added to the cell extract, and DNA was sheared by sonication to an average size of ∼500 bp. Insoluble cellular material was removed by microcentrifugation for 10 min, and the supernatant was transferred to a fresh tube. Proteins were immunoprecipitated by diluting a fraction of the cross-linked cell extract with IP buffer to a final volume of 800 μL. This was then incubated with 20 μL of Protein A-Sepharose beads (Amersham-Pharmacia) and either no antibody, RNAP β subunit mouse monoclonal (NeoClone), LexA rabbit polyclonal antibody (Upstate), or Gal4 DNA-binding domain rabbit polyclonal antibody (Upstate) for 90 min at room temperature with gentle mixing. Samples were then washed twice with IP buffer, once with IP buffer + 500 mM NaCl, once with wash buffer (10 mM Tris-HCl at pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet-P40, 0.5% sodium deoxycholate), and once with TE (pH 7.5). Immunoprecipitated complexes were eluted by incubation of beads with elution buffer (50 mM Tris-HCl at pH 7.5, 10 mM EDTA, 1% SDS) at 65°C for 10 min. Immunoprecipitated samples and the corresponding “input” sample were decross-linked by incubation for 2 h at 42°C and for 6 h at 65°C in 0.5× elution buffer + 0.8 mg/mL Pronase. DNA was purified using a PCR purification kit (QIAGEN). All ChIPs were performed at least twice.

Transcription Factor Binding Sites


TACTGTACATCCATACAGTA
TACTGTTTATTTATACAGTA
TACTGTATATAAAAACAGTA
AACTGTATATAAATACAGTT
TACTGTATATAAAACCAGTT
TACTGTATAAAATCACAGTT
TGCTGTATATACTCACAGCA
ACCTGTATAAATAACCAGTA
TACTGTACGTATCGACAGTT
TACTGATGATATATACAGGT
TACTGTATATAAAAACAGTA
TACTGTATGCTCATACAGTA
ACCTGAATGAATATACAGTA
AACTGGACGTTTGTACAGCA
AACTGTTTTTTTATCCAGTA
AACTGGATAAAATTACAGGG
TACTGTATGATTATCCAGTT
CACTGTATACTTTACCAGTG
TCCTGTTAATCCATACAGCA
CACTGGATAGATAACCAGCA
GACTGTATAAAACCACAGCC
CGCTGGATATCTATCCAGCA
TATTGGCTGTTTATACAGTA
ATCTGTATATATACCCAGCT
TACTGGATATTTAAACAGGT
TACTGGATAACACACCATGA
GCATGTTGAAATAGCCAGTA
AAATGTTTTTACATCCACTA
AACTCTATATTACCCCAGTT
TACTGTACATCCATACAGTA
TACTGTTTATTTATACAGTA
TACTGTATATAAAAACAGTA
AACTGTATATAAATACAGTT
TACTGTATATAAAACCAGTT
TACTGTATAAAATCACAGTT
TGCTGTATATACTCACAGCA
ACCTGTATAAATAACCAGTA
TACTGTACGTATCGACAGTT
TACTGATGATATATACAGGT
TACTGTATATAAAAACAGTA
TACTGTATGCTCATACAGTA
ACCTGAATGAATATACAGTA
AACTGGACGTTTGTACAGCA
AACTGTTTTTTTATCCAGTA
AACTGGATAAAATTACAGGG
TACTGTATGATTATCCAGTT
CACTGTATACTTTACCAGTG
TCCTGTTAATCCATACAGCA
CACTGGATAGATAACCAGCA
GACTGTATAAAACCACAGCC
CGCTGGATATCTATCCAGCA
TATTGGCTGTTTATACAGTA
ATCTGTATATATACCCAGCT
TACTGGATATTTAAACAGGT
TACTGGATAACACACCATGA
GCATGTTGAAATAGCCAGTA
AAATGTTTTTACATCCACTA
AACTCTATATTACCCCAGTT

Gene Regulation

Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.

Site sequence Regulated genes Gene diagram Experimental techniques TF function TF type
TACTGTACATCCATACAGTA
... ... yccF yccS sxy sulA
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TACTGTTTATTTATACAGTA
... ... istR tisB
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TACTGTATATAAAAACAGTA
... ... dsbB umuC umuD hlyE
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
AACTGTATATAAATACAGTT
... ... dinD yicC
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TACTGTATATAAAACCAGTT
... ... recN nadK
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TACTGTATAAAATCACAGTT
... ... yebG purT
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TGCTGTATATACTCACAGCA
... ... dinF lexA dgkA
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
ACCTGTATAAATAACCAGTA
... ... dinI pyrC
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TACTGTACGTATCGACAGTT
... ... ydjM yniC
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TACTGATGATATATACAGGT
... ... hsdM hsdS symE symR
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TACTGTATATAAAAACAGTA
... ... dsbB umuC umuD hlyE
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TACTGTATGCTCATACAGTA
... ... recA ygaD
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
ACCTGAATGAATATACAGTA
... ... ssb yjbQ yjbR uvrA
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
AACTGGACGTTTGTACAGCA
... ... udp rmuC
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
AACTGTTTTTTTATCCAGTA
... ... bioB bioF bioC bioD uvrB
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
AACTGGATAAAATTACAGGG
... ... yehI yehH metG
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TACTGTATGATTATCCAGTT
... ... arsR dinQ
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
CACTGTATACTTTACCAGTG
... ... dinB lfhA lafU
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TCCTGTTAATCCATACAGCA
... ... lrp ftsK
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
CACTGGATAGATAACCAGCA
... ... ves cho nadE
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
GACTGTATAAAACCACAGCC
... ... araD polB
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
CGCTGGATATCTATCCAGCA
... ... yobI ruvB ruvA
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TATTGGCTGTTTATACAGTA
... ... dinG ybiA ybiB
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
ATCTGTATATATACCCAGCT
... ... yifL uvrD dapF yigA xerC yigB
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TACTGGATATTTAAACAGGT
... ... ycgJ minE minD minC
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
TACTGGATAACACACCATGA
... ... yoaC yoaB
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
GCATGTTGAAATAGCCAGTA
... ... gpp rep ppiC yifN
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
AAATGTTTTTACATCCACTA
... ... lpcA ivy fadE
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer
AACTCTATATTACCCCAGTT
... ... ydeR ydeS ydeP ydeQ ydeT
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details PSSM site search (ECO:0005659) - repressor dimer