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Escherichia coli K-12 substr. MG1655 Polypeptide: LacI DNA-binding transcriptional repressor



Gene: lacI Accession Numbers: EG10525 (EcoCyc), b0345, ECK0342

Synonyms: transcriptional repressor of the lac operon

Regulation Summary Diagram: ?

Component of: LacI-allolactose

Summary:
The Lactose inhibitor," LacI, is a DNA-binding transcription factor that represses transcription of the operon involved in transport and catabolism of lactose [Hudson90, Vossen96, Lewis05]. In the absence of allolactose, LacI represses the lac operon by preventing open promoter complex formation for transcription [Hudson90, Sanchez11]. In this repression system, LacI binds to two operators, and formation of the repressor loop is critical [Fried96, Perros96, Balaeff04, Hudson90]. This repressor binds in tandem to inverted repeat sequences that are 21 nucleotides long and possess conserved motifs [Hudson90, Fried96, Perros96].

LacI is negatively autoregulated when it binds to two DNA-binding sites, one located downstream of the lacI gene and the other one located in the coding sequence for the C terminus of LacI. The protein when bound to these sites forms a loop that inhibits the transcription elongation, thus producing truncated proteins that are tagged for degradation by the small peptide SsrA [Abo00, Semsey13].

Induction occurs when the physiological inducer, allolactose, binds to the lac repressor, preventing it from binding to the operator [Hudson90, Lewis05]. Nonphysiological analogs, such as thiogalactosides, can function as inducers too. A single mutation, W220F, in the inducer-binding site of the LacI repressor reduces leakiness [Mooney13].

The LacI protein belongs to the GalR/LacI family and as a member of this family of transcriptional regulators, LacI contains three domains: a helix-turn-helix motif located in the N terminus, the central domain that binds to LacI sugar ligands, and the C terminal, which comprises the tetramerization domain[Lewis05].

The crystal structure of the LacI binding site for allosteric effectors has been resolved [Daber07]. The importance of the N125 and D149 residues of the N terminal in the recognition of the sugar effector binding site has been determined [Daber07, Xu11].

Based on a computational rod model of the DNA-LacI complex, the looping of both linear DNA and supercoiled DNA minicircles over a broad range of DNA interoperator lengths was analyzed. The analysis showed that the most stable loops for linear DNA occur when LacI adopts the extended conformation [Hirsh11]. On the other hand, in an in vivo single-molecule assay, the sliding process for the LacI TF was determined. LacI slides 45 +/- 10 bp on chromosomal DNA, and this can be obstructed by other DNA-bound proteins near the operator. LacI slides over its natural lacO1 operator several times before binding (>90% repressor frequency), suggesting a trade-off between a rapid search on nonspecific sequences and fast binding at the specific sequence [Hammar12].

Citations: [Platt73, Ganem73, Beyreuther75, Farabaugh78, Gordon88, Boelens88, Lamerichs89, Lehming90, KistersWoike91, Markiewicz94, Jacob61, PARDEE59]

Locations: cytosol

Map Position: [365,652 <- 366,734] (7.88 centisomes)
Length: 1083 bp / 360 aa

Molecular Weight of Polypeptide: 38.59 kD (from nucleotide sequence)

pI: 6.7

Unification Links: ASAP:ABE-0001189 , CGSC:578 , DIP:DIP-10079N , DisProt:DP00433 , EchoBASE:EB0520 , EcoGene:EG10525 , EcoliWiki:b0345 , Mint:MINT-6478062 , ModBase:P03023 , OU-Microarray:b0345 , PortEco:lacI , PR:PRO_000023070 , Pride:P03023 , Protein Model Portal:P03023 , RefSeq:NP_414879 , RegulonDB:EG10525 , SMR:P03023 , String:511145.b0345 , UniProt:P03023

Relationship Links: InterPro:IN-FAMILY:IPR000843 , InterPro:IN-FAMILY:IPR010982 , InterPro:IN-FAMILY:IPR026908 , InterPro:IN-FAMILY:IPR028082 , Panther:IN-FAMILY:PTHR30146:SF1 , PDB:Structure:1CJG , PDB:Structure:1EFA , PDB:Structure:1JWL , PDB:Structure:1JYE , PDB:Structure:1JYF , PDB:Structure:1L1M , PDB:Structure:1LBG , PDB:Structure:1LBH , PDB:Structure:1LBI , PDB:Structure:1LCC , PDB:Structure:1LCD , PDB:Structure:1LQC , PDB:Structure:1LTP , PDB:Structure:1OSL , PDB:Structure:1TLF , PDB:Structure:1Z04 , PDB:Structure:2BJC , PDB:Structure:2KEI , PDB:Structure:2KEJ , PDB:Structure:2KEK , PDB:Structure:2P9H , PDB:Structure:2PAF , PDB:Structure:2PE5 , PDB:Structure:3EDC , Pfam:IN-FAMILY:PF00356 , Prints:IN-FAMILY:PR00036 , Prosite:IN-FAMILY:PS00356 , Prosite:IN-FAMILY:PS50932 , Smart:IN-FAMILY:SM00354

In Paralogous Gene Group: 30 (25 members)

In Reactions of unknown directionality:

Not in pathways:
LacI + allolactose = LacI-allolactose

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0045892 - negative regulation of transcription, DNA-templated Inferred from experiment [MullerHill68]
GO:0006351 - transcription, DNA-templated Inferred by computational analysis [UniProtGOA11]
GO:0006355 - regulation of transcription, DNA-templated Inferred by computational analysis [UniProtGOA11, GOA01]
Molecular Function: GO:0000986 - bacterial-type RNA polymerase core promoter proximal region sequence-specific DNA binding Inferred from experiment [Riggs70]
GO:0001141 - bacterial-type RNA polymerase core promoter proximal region sequence-specific DNA binding transcription factor activity involved in negative regulation of transcription Inferred from experiment [Lehming87]
GO:0003677 - DNA binding Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0003700 - sequence-specific DNA binding transcription factor activity Inferred by computational analysis [GOA01]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Lasserre06]

MultiFun Terms: information transfer RNA related Transcription related
metabolism carbon utilization carbon compounds
regulation genetic unit regulated operon
regulation type of regulation transcriptional level repressor

DNA binding site length: 21 base-pairs

Symmetry: Inverted Repeat

Consensus DNA Binding Sequence: AATTGTGAGCGGctcaCAATt

Regulated Transcription Units (1 total): ?

Notes:

Essentiality data for lacI 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]

Subunit of: LacI-allolactose

Synonyms: B0345, LacI

Subunit composition of LacI-allolactose = [LacI][allolactose]
         LacI DNA-binding transcriptional repressor = LacI (extended summary available)

Sequence Length: 360 AAs

Molecular Weight: 38.59 kD (from nucleotide sequence)

Relationship Links: PDB:Structure:1LBI , Pfam:IN-FAMILY:PF00356

In Reactions of unknown directionality:

Not in pathways:
LacI + allolactose = LacI-allolactose

MultiFun Terms: information transfer RNA related Transcription related
metabolism carbon utilization carbon compounds
regulation genetic unit regulated operon
regulation type of regulation transcriptional level repressor


Sequence Features

Feature Class Location Citations Comment
Conserved-Region 1 -> 58
[UniProt09]
UniProt: HTH lacI-type;
DNA-Binding-Region 6 -> 25
[UniProt10a]
UniProt: H-T-H motif;
Mutagenesis-Variant 17
[UniProt10a]
Alternate sequence: Y → H; UniProt: Broadening of specificity;
Mutagenesis-Variant 22
[UniProt10a]
Alternate sequence: R → N; UniProt: Recognizes an operator variant;
Extrinsic-Sequence-Variant 282
[UniProt10a]
Alternate sequence: Y → D; UniProt: (in T41 mutant);
Sequence-Conflict 286
[Chung97, Beyreuther75, Farabaugh78, UniProt10a]
Alternate sequence: L → S; UniProt: (in Ref. 1, 4 and 7);


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
10/20/97 Gene b0345 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10525; confirmed by SwissProt match.


References

Abo00: Abo T, Inada T, Ogawa K, Aiba H (2000). "SsrA-mediated tagging and proteolysis of LacI and its role in the regulation of lac operon." EMBO J 19(14);3762-9. PMID: 10899129

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

Balaeff04: Balaeff A, Mahadevan L, Schulten K (2004). "Structural basis for cooperative DNA binding by CAP and lac repressor." Structure 12(1);123-32. PMID: 14725772

Beyreuther75: Beyreuther K, Adler K, Fanning E, Murray C, Klemm A, Geisler N (1975). "Amino-acid sequence of lac repressor from Escherichia coli. Isolation, sequence analysis and sequence assembly of tryptic peptides and cyanogen-bromide fragments." Eur J Biochem 1975;59(2);491-509. PMID: 1107032

Boelens88: Boelens R, Lamerichs RM, Rullmann JA, van Boom JH, Kaptein R (1988). "The interaction of lac repressor headpiece with its operator: an NMR view." Protein Seq Data Anal 1988;1(6);487-98. PMID: 3064080

Chung97: Chung E., Allen E., Araujo R., Aparicio A.M., Davis K., Duncan M., Federspiel N., Hyman R., Kalman S., Komp C., Kurdi O., Lew H., Lin D., Namath A., Oefner P., Roberts D., Schramm S., Davis R.W. (1997). "Sequence of minutes 4-25 of Escherichia coli." Data submission to EMBL/GenBank/DDBJ databases on 1997-01.

Daber07: Daber R, Stayrook S, Rosenberg A, Lewis M (2007). "Structural analysis of lac repressor bound to allosteric effectors." J Mol Biol 370(4);609-19. PMID: 17543986

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

Farabaugh78: Farabaugh PJ (1978). "Sequence of the lacI gene." Nature 1978;274(5673);765-9. PMID: 355891

Fried96: Fried MG, Hudson JM (1996). "DNA looping and lac repressor-CAP interaction." Science 274(5294);1930-1; author reply 1931-2. PMID: 8984648

Ganem73: Ganem D, Miller JH, Files JG, Platt T, Weber K (1973). "Reinitiation of a lac repressor fragment at a codon other than AUG." Proc Natl Acad Sci U S A 1973;70(11);3165-9. PMID: 4594037

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

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

Gordon88: Gordon AJ, Burns PA, Fix DF, Yatagai F, Allen FL, Horsfall MJ, Halliday JA, Gray J, Bernelot-Moens C, Glickman BW (1988). "Missense mutation in the lacI gene of Escherichia coli. Inferences on the structure of the repressor protein." J Mol Biol 1988;200(2);239-51. PMID: 3286877

Hammar12: Hammar P, Leroy P, Mahmutovic A, Marklund EG, Berg OG, Elf J (2012). "The lac repressor displays facilitated diffusion in living cells." Science 336(6088);1595-8. PMID: 22723426

Hirsh11: Hirsh AD, Lillian TD, Lionberger TA, Perkins NC (2011). "DNA modeling reveals an extended lac repressor conformation in classic in vitro binding assays." Biophys J 101(3);718-26. PMID: 21806940

Hudson90: Hudson JM, Fried MG (1990). "Co-operative interactions between the catabolite gene activator protein and the lac repressor at the lactose promoter." J Mol Biol 214(2);381-96. PMID: 2166165

Jacob61: Jacob F, Monod J (1961). "Genetic regulatory mechanisms in the synthesis of proteins." J Mol Biol 3;318-56. PMID: 13718526

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

KistersWoike91: Kisters-Woike B, Lehming N, Sartorius J, von Wilcken-Bergmann B, Muller-Hill B (1991). "A model of the lac repressor-operator complex based on physical and genetic data." Eur J Biochem 1991;198(2);411-9. PMID: 2040302

Lamerichs89: Lamerichs RM, Boelens R, van der Marel GA, van Boom JH, Kaptein R, Buck F, Fera B, Ruterjans H (1989). "H NMR study of a complex between the lac repressor headpiece and a 22 base pair symmetric lac operator." Biochemistry 1989;28(7);2985-91. PMID: 2742823

Lasserre06: Lasserre JP, Beyne E, Pyndiah S, Lapaillerie D, Claverol S, Bonneu M (2006). "A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis." Electrophoresis 27(16);3306-21. PMID: 16858726

Lehming87: Lehming N, Sartorius J, Niemoller M, Genenger G, v Wilcken-Bergmann B, Muller-Hill B (1987). "The interaction of the recognition helix of lac repressor with lac operator." EMBO J 6(10);3145-53. PMID: 2826131

Lehming90: Lehming N, Sartorius J, Kisters-Woike B, von Wilcken-Bergmann B, Muller-Hill B (1990). "Mutant lac repressors with new specificities hint at rules for protein--DNA recognition." EMBO J 1990;9(3);615-21. PMID: 2178920

Lewis05: Lewis M (2005). "The lac repressor." C R Biol 328(6);521-48. PMID: 15950160

Markiewicz94: Markiewicz P, Kleina LG, Cruz C, Ehret S, Miller JH (1994). "Genetic studies of the lac repressor. XIV. Analysis of 4000 altered Escherichia coli lac repressors reveals essential and non-essential residues, as well as "spacers" which do not require a specific sequence." J Mol Biol 1994;240(5);421-33. PMID: 8046748

Mooney13: Mooney RA, Landick R (2013). "Building a better stop sign: understanding the signals that terminate transcription." Nat Methods 10(7);618-9. PMID: 23807194

MullerHill68: Muller-Hill B, Crapo L, Gilbert W (1968). "Mutants that make more lac repressor." Proc Natl Acad Sci U S A 59(4);1259-64. PMID: 4870861

PARDEE59: PARDEE AB, PRESTIDGE LS (1959). "On the nature of the repressor of beta-galactosidase synthesis in Escherichia coli." Biochim Biophys Acta 36;545-7. PMID: 14430363

Perros96: Perros M, Steitz TA (1996). "DNA looping and lac repressor-CAP interaction." Science 274(5294);1929-30; author reply 1931-2. PMID: 8984647

Platt73: Platt T, Files JG, Weber K (1973). "Lac repressor. Specific proteolytic destruction of the NH 2 -terminal region and loss of the deoxyribonucleic acid-binding activity." J Biol Chem 1973;248(1);110-21. PMID: 4571224

Riggs70: Riggs AD, Suzuki H, Bourgeois S (1970). "Lac repressor-operator interaction. I. Equilibrium studies." J Mol Biol 48(1);67-83. PMID: 4915295

Sanchez11: Sanchez A, Osborne ML, Friedman LJ, Kondev J, Gelles J (2011). "Mechanism of transcriptional repression at a bacterial promoter by analysis of single molecules." EMBO J 30(19);3940-6. PMID: 21829165

Semsey13: Semsey S, Jauffred L, Csiszovszki Z, Erdossy J, Steger V, Hansen S, Krishna S (2013). "The effect of LacI autoregulation on the performance of the lactose utilization system in Escherichia coli." Nucleic Acids Res 41(13);6381-90. PMID: 23658223

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

Vossen96: Vossen KM, Stickle DF, Fried MG (1996). "The mechanism of CAP-lac repressor binding cooperativity at the E. coli lactose promoter." J Mol Biol 255(1);44-54. PMID: 8568874

Xu11: Xu J, Liu S, Chen M, Ma J, Matthews KS (2011). "Altering residues N125 and D149 impacts sugar effector binding and allosteric parameters in Escherichia coli lactose repressor." Biochemistry 50(42);9002-13. PMID: 21928765

Other References Related to Gene Regulation

Manso11: Manso I, Garcia JL, Galan B (2011). "Escherichia coli mhpR gene expression is regulated by catabolite repression mediated by the cAMP-CRP complex." Microbiology 157(Pt 2);593-600. PMID: 20966094

Quan12: Quan S, Ray JC, Kwota Z, Duong T, Balazsi G, Cooper TF, Monds RD (2012). "Adaptive evolution of the lactose utilization network in experimentally evolved populations of Escherichia coli." PLoS Genet 8(1);e1002444. PMID: 22253602

Steege77: Steege DA (1977). "5'-Terminal nucleotide sequence of Escherichia coli lactose repressor mRNA: features of translational initiation and reinitiation sites." Proc Natl Acad Sci U S A 1977;74(10);4163-7. PMID: 337294

Torres03: Torres B, Porras G, Garcia JL, Diaz E (2003). "Regulation of the mhp cluster responsible for 3-(3-hydroxyphenyl) propionic acid degradation in escherichia coli." J Biol Chem 278(30);27575-85. PMID: 12748194


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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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