Escherichia coli K-12 substr. MG1655 Polypeptide: Rob DNA-binding transcriptional activator

Gene: rob Accession Numbers: EG11366 (EcoCyc), b4396, ECK4388

Synonyms: cbpB, robA, right origin-binding protein

Regulation Summary Diagram: ?

Regulation summary diagram for rob

Rob is a transcriptional dual regulator. Its N-terminal domain shares 49% identity with MarA and SoxS [Cohen93]. These proteins activate a common set of about 50 target genes [Barbosa00, Martin02, Martin03, Pomposiello01], the marA/soxS/rob regulon, involved in antibiotic resistance [Martin96, Griffith05, Ariza95], superoxide resistance [Jair96, Nunoshiba92, Wu92b], and tolerance to organic solvents [White97, Aono98] and heavy metals [Nakajima95]. The activity of each protein is induced by different signals: the activity of Rob is increased with dipyridyl, bile salts, or decanoate [Rosner02, Rosenberg03], and the activities of MarA and SoxS are increased by the aromatic weak acid salicylate [Pomposiello01] and oxidative stress [Demple96], respectively. Cross talk between the mar and rob systems plays an important role in the response to salicylate [Chubiz12].

Many genes are regulated by all three proteins; however, some genes are regulated by only one of them. The differential regulation of these genes might be caused by the degeneracy of their DNA-binding sites [Pomposiello03].

Rob, MarA, and SoxS bind as monomers to the same DNA site, a degenerate 20-bp sequence known as the Mar-Sox-Rob box, which has to be in a specific orientation and distance relative to the -35 and -10 boxes of the promoter [Martin99, Wood99a]. In class I promoters, the activators bind upstream of the -35 box and are generally oriented in the backward direction, while in class II promoters the proteins overlap the -35 promoter hexamer and generally are oriented in the forward direction [Martin99, Wood99a, Taliaferro12]. As a subset of the class I promoters, the Mar-Sox-Rob box is separated by ~30 bp from the -10 hexamer but can be functional in either orientation [Martin99, Wood99a].

The Mar-Sox-Rob box contains an "invariant A" at position 1, two recognition elements, the RE1 at position 4-7 and RE2 at position 15-18, and a 7-bp A/T-rich spacer separating these elements [Kwon00, Dangi01, Griffith01]. There are more than 10,000 such binding sites per genome [Griffith02]. However, the majority of these sites are not functional because they are not in the proper orientation or distance relative to the promoter [Martin02a]. It was proposed that these proteins bind to their target sites by a mechanism named "DNA scanning" or "prerecruitment." In prerecruitment, the protein first binds to RNA polymerase in solution, and the binary complex then scans the DNA to find its binding sites [Martin02a, Griffith02].

Rob consists of two domains and belongs to the AraC/XylS family [Gallegos97]. The N-terminal domain is the DNA-binding domain and is homologous to MarA and SoxS and the C-terminal domain of AraC. It carries two helix-turn-helix (HTH) motifs for DNA binding. One of them, located in the N-terminal region, interacts with the element RE1 of the Mar-Sox-Rob box, and the HTH located in the C-terminal region interacts with RE2 [Griffith02a, Rhee98, Dangi01]. In the case of Rob, it appears that only one of the two HTH motifs makes base-specific contacts with DNA [Kwon00]. The crystal structures of Rob [Kwon00] and MarA [Rhee98] in complex with DNA and the solution structure of the DNA-binding domain of AraC [Rodgers09] have been solved.

The C-terminal domain of Rob is similar to a portion of the enzyme GalT [Kwon00]. It regulates the activity of Rob by a "sequestration-disposal" mechanism, that is, it mediates the sequestration of Rob into intracellular foci. Sequestered Rob is inactive. Upon addition of the inducer, Rob is released in its active form [Griffith09]. The C-terminal domain blocks in addition proteolytic degradation by Lon protease [Griffith09].

rob appears to be transcribed abundantly at 5,000-10,000 molecules per cell [Skarstad93, Ali99], throughout the growth cycle. rob expression is enhanced at the stationary phase and under glucose and phosphate starvation, and it was suggested that expression of rob is σS dependent [Kakeda95].

Reviews: [Alekshun99, Demple96, Randall02].

Locations: cytosol

Map Position: [4,632,464 <- 4,633,333] (99.84 centisomes, 359°)
Length: 870 bp / 289 aa

Molecular Weight of Polypeptide: 33.145 kD (from nucleotide sequence)

pI: 7.0

Unification Links: ASAP:ABE-0014420 , CGSC:34661 , DIP:DIP-47868N , EchoBASE:EB1340 , EcoGene:EG11366 , EcoliWiki:b4396 , Mint:MINT-1246646 , ModBase:P0ACI0 , OU-Microarray:b4396 , PortEco:rob , PR:PRO_000023802 , Pride:P0ACI0 , Protein Model Portal:P0ACI0 , RefSeq:NP_418813 , RegulonDB:EG11366 , SMR:P0ACI0 , String:511145.b4396 , UniProt:P0ACI0

Relationship Links: InterPro:IN-FAMILY:IPR009057 , InterPro:IN-FAMILY:IPR010499 , InterPro:IN-FAMILY:IPR011256 , InterPro:IN-FAMILY:IPR018060 , InterPro:IN-FAMILY:IPR018062 , InterPro:IN-FAMILY:IPR020449 , InterPro:IN-FAMILY:IPR029442 , PDB:Structure:1D5Y , Pfam:IN-FAMILY:PF00165 , Pfam:IN-FAMILY:PF06445 , Prints:IN-FAMILY:PR00032 , Prosite:IN-FAMILY:PS00041 , Prosite:IN-FAMILY:PS01124 , Smart:IN-FAMILY:SM00342 , Smart:IN-FAMILY:SM00871

In Paralogous Gene Group: 22 (29 members)

Genetic Regulation Schematic: ?

Genetic regulation schematic for rob

GO Terms:

Biological Process: GO:0006355 - regulation of transcription, DNA-templated Inferred by computational analysis [GOA01a]
Molecular Function: GO:0043565 - sequence-specific DNA binding Inferred from experiment Inferred by computational analysis [GOA01a, Azam99]
GO:0003677 - DNA binding Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0003700 - sequence-specific DNA binding transcription factor activity Inferred by computational analysis [GOA01a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm

MultiFun Terms: information transfer DNA related DNA replication
information transfer protein related nucleoproteins, basic proteins
information transfer RNA related Transcription related
regulation genetic unit regulated regulon
regulation type of regulation transcriptional level activator

DNA binding site length: 20 base-pairs

Symmetry: Asymmetric

Consensus DNA Binding Sequence: tAcgGCAcgaattGtcAAgn

Regulated Transcription Units (18 total): ?


Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Essentiality data for rob knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]

Last-Curated ? 20-Mar-2009 by Gama-Castro S , UNAM

Sequence Features

Protein sequence of Rob DNA-binding transcriptional activator with features indicated

Feature Class Location Citations Comment
DNA-Binding-Region 24 -> 43
UniProt: H-T-H motif; Non-Experimental Qualifier: by similarity;

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


10/20/97 Gene b4396 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11366; confirmed by SwissProt match.


Alekshun99: Alekshun MN, Levy SB (1999). "The mar regulon: multiple resistance to antibiotics and other toxic chemicals." Trends Microbiol 7(10);410-3. PMID: 10498949

Ali99: Ali Azam T, Iwata A, Nishimura A, Ueda S, Ishihama A (1999). "Growth phase-dependent variation in protein composition of the Escherichia coli nucleoid." J Bacteriol 181(20);6361-70. PMID: 10515926

Aono98: Aono R (1998). "Improvement of organic solvent tolerance level of Escherichia coli by overexpression of stress-responsive genes." Extremophiles 2(3);239-48. PMID: 9783171

Ariza95: Ariza RR, Li Z, Ringstad N, Demple B (1995). "Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein." J Bacteriol 1995;177(7);1655-61. PMID: 7896685

Azam99: Azam TA, Ishihama A (1999). "Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity." J Biol Chem 274(46);33105-13. PMID: 10551881

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Barbosa00: Barbosa TM, Levy SB (2000). "Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA." J Bacteriol 182(12);3467-74. PMID: 10852879

Chubiz12: Chubiz LM, Glekas GD, Rao CV (2012). "Transcriptional cross talk within the mar-sox-rob regulon in Escherichia coli is limited to the rob and marRAB operons." J Bacteriol 194(18);4867-75. PMID: 22753060

Cohen93: Cohen SP, Hachler H, Levy SB (1993). "Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli." J Bacteriol 1993;175(5);1484-92. PMID: 8383113

Dangi01: Dangi B, Pelupessey P, Martin RG, Rosner JL, Louis JM, Gronenborn AM (2001). "Structure and dynamics of MarA-DNA complexes: an NMR investigation." J Mol Biol 314(1);113-27. PMID: 11724537

Demple96: Demple B (1996). "Redox signaling and gene control in the Escherichia coli soxRS oxidative stress regulon--a review." Gene 179(1);53-7. PMID: 8955629

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Gallegos97: Gallegos MT, Schleif R, Bairoch A, Hofmann K, Ramos JL (1997). "Arac/XylS family of transcriptional regulators." Microbiol Mol Biol Rev 61(4);393-410. PMID: 9409145

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

Griffith01: Griffith KL, Wolf RE (2001). "Systematic mutagenesis of the DNA binding sites for SoxS in the Escherichia coli zwf and fpr promoters: identifying nucleotides required for DNA binding and transcription activation." Mol Microbiol 40(5);1141-54. PMID: 11401718

Griffith02: Griffith KL, Shah IM, Myers TE, O'Neill MC, Wolf RE (2002). "Evidence for "pre-recruitment" as a new mechanism of transcription activation in Escherichia coli: the large excess of SoxS binding sites per cell relative to the number of SoxS molecules per cell." Biochem Biophys Res Commun 291(4);979-86. PMID: 11866462

Griffith02a: Griffith KL, Wolf RE (2002). "A comprehensive alanine scanning mutagenesis of the Escherichia coli transcriptional activator SoxS: identifying amino acids important for DNA binding and transcription activation." J Mol Biol 322(2);237-57. PMID: 12217688

Griffith05: Griffith KL, Becker SM, Wolf RE (2005). "Characterization of TetD as a transcriptional activator of a subset of genes of the Escherichia coli SoxS/MarA/Rob regulon." Mol Microbiol 56(4);1103-17. PMID: 15853893

Griffith09: Griffith KL, Fitzpatrick MM, Keen EF, Wolf RE (2009). "Two functions of the C-terminal domain of Escherichia coli Rob: mediating "sequestration-dispersal" as a novel off-on switch for regulating Rob's activity as a transcription activator and preventing degradation of Rob by Lon protease." J Mol Biol 388(3);415-30. PMID: 19289129

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Jair96: Jair KW, Yu X, Skarstad K, Thony B, Fujita N, Ishihama A, Wolf RE (1996). "Transcriptional activation of promoters of the superoxide and multiple antibiotic resistance regulons by Rob, a binding protein of the Escherichia coli origin of chromosomal replication." J Bacteriol 1996;178(9);2507-13. PMID: 8626315

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Kakeda95: Kakeda M, Ueguchi C, Yamada H, Mizuno T (1995). "An Escherichia coli curved DNA-binding protein whose expression is affected by the stationary phase-specific sigma factor sigma S." Mol Gen Genet 248(5);629-34. PMID: 7476863

Kwon00: Kwon HJ, Bennik MH, Demple B, Ellenberger T (2000). "Crystal structure of the Escherichia coli Rob transcription factor in complex with DNA." Nat Struct Biol 7(5);424-30. PMID: 10802742

Martin02: Martin RG, Rosner JL (2002). "Genomics of the marA/soxS/rob regulon of Escherichia coli: identification of directly activated promoters by application of molecular genetics and informatics to microarray data." Mol Microbiol 44(6);1611-24. PMID: 12067348

Martin02a: Martin RG, Gillette WK, Martin NI, Rosner JL (2002). "Complex formation between activator and RNA polymerase as the basis for transcriptional activation by MarA and SoxS in Escherichia coli." Mol Microbiol 43(2);355-70. PMID: 11985714

Martin03: Martin RG, Rosner JL (2003). "Analysis of microarray data for the marA, soxS, and rob regulons of Escherichia coli." Methods Enzymol 370;278-80. PMID: 14712652

Martin96: Martin RG, Jair KW, Wolf RE, Rosner JL (1996). "Autoactivation of the marRAB multiple antibiotic resistance operon by the MarA transcriptional activator in Escherichia coli." J Bacteriol 1996;178(8);2216-23. PMID: 8636021

Martin99: Martin RG, Gillette WK, Rhee S, Rosner JL (1999). "Structural requirements for marbox function in transcriptional activation of mar/sox/rob regulon promoters in Escherichia coli: sequence, orientation and spatial relationship to the core promoter." Mol Microbiol 1999;34(3);431-41. PMID: 10564485

Nakajima95: Nakajima H, Kobayashi K, Kobayashi M, Asako H, Aono R (1995). "Overexpression of the robA gene increases organic solvent tolerance and multiple antibiotic and heavy metal ion resistance in Escherichia coli." Appl Environ Microbiol 61(6);2302-7. PMID: 7793951

Nunoshiba92: Nunoshiba T, Hidalgo E, Amabile Cuevas CF, Demple B (1992). "Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene." J Bacteriol 1992;174(19);6054-60. PMID: 1400156

Pomposiello01: Pomposiello PJ, Bennik MH, Demple B (2001). "Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate." J Bacteriol 183(13);3890-902. PMID: 11395452

Pomposiello03: Pomposiello PJ, Koutsolioutsou A, Carrasco D, Demple B (2003). "SoxRS-regulated expression and genetic analysis of the yggX gene of Escherichia coli." J Bacteriol 185(22);6624-32. PMID: 14594836

Randall02: Randall LP, Woodward MJ (2002). "The multiple antibiotic resistance (mar) locus and its significance." Res Vet Sci 72(2);87-93. PMID: 12027588

Rhee98: Rhee S, Martin RG, Rosner JL, Davies DR (1998). "A novel DNA-binding motif in MarA: the first structure for an AraC family transcriptional activator." Proc Natl Acad Sci U S A 95(18);10413-8. PMID: 9724717

Rodgers09: Rodgers ME, Schleif R (2009). "Solution structure of the DNA binding domain of AraC protein." Proteins 77(1);202-8. PMID: 19422057

Rosenberg03: Rosenberg EY, Bertenthal D, Nilles ML, Bertrand KP, Nikaido H (2003). "Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein." Mol Microbiol 48(6);1609-19. PMID: 12791142

Rosner02: Rosner JL, Dangi B, Gronenborn AM, Martin RG (2002). "Posttranscriptional activation of the transcriptional activator Rob by dipyridyl in Escherichia coli." J Bacteriol 184(5);1407-16. PMID: 11844771

Skarstad93: Skarstad K, Thony B, Hwang DS, Kornberg A (1993). "A novel binding protein of the origin of the Escherichia coli chromosome." J Biol Chem 268(8);5365-70. PMID: 8449900

Taliaferro12: Taliaferro LP, Keen EF, Sanchez-Alberola N, Wolf RE (2012). "Transcription Activation by Escherichia coli Rob at Class II Promoters: Protein-Protein Interactions between Rob's N-Terminal Domain and the σ(70) Subunit of RNA Polymerase." J Mol Biol 419(3-4);139-57. PMID: 22465792

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

White97: White DG, Goldman JD, Demple B, Levy SB (1997). "Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, or robA in Escherichia coli." J Bacteriol 179(19);6122-6. PMID: 9324261

Wood99a: Wood TI, Griffith KL, Fawcett WP, Jair KW, Schneider TD, Wolf RE (1999). "Interdependence of the position and orientation of SoxS binding sites in the transcriptional activation of the class I subset of Escherichia coli superoxide-inducible promoters." Mol Microbiol 34(3);414-30. PMID: 10564484

Wu92b: Wu J, Weiss B (1992). "Two-stage induction of the soxRS (superoxide response) regulon of Escherichia coli." J Bacteriol 174(12);3915-20. PMID: 1317841

Other References Related to Gene Regulation

McMurry10: McMurry LM, Levy SB (2010). "Evidence that regulatory protein MarA of Escherichia coli represses rob by steric hindrance." J Bacteriol 192(15);3977-82. PMID: 20453091

Schneiders06: Schneiders T, Levy SB (2006). "MarA mediated transcriptional repression of the rob promoter." J Biol Chem 281(15):10049-51. PMID: 16478729

Viveiros07: Viveiros M, Dupont M, Rodrigues L, Couto I, Davin-Regli A, Martins M, Pages JM, Amaral L (2007). "Antibiotic stress, genetic response and altered permeability of E. coli." PLoS ONE 2;e365. PMID: 17426813

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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