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Nucleic Acids Res. 1998 August 15; 26(16): 3700–3706.
PMCID: PMC147751

Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli.


This report describes an Escherichia coli genetic system that permits bacterial genetic methods to be applied to the study of essentially any prokaryotic or eukaryotic site-specific DNA binding protein. It consists of two parts. The first part is a set of tools that facilitate construction of customized E.coli strains bearing single copy lacZYA reporters that are repressed by a specific target protein. The second part is a pair of regulatable protein expression vectors that permit in vivo production of the target protein at levels appropriate for genetic experiments. When expressed in a properly designed reporter strain, the target protein represses the lac genes, resulting in an E.coli phenotype that can be quantitatively measured or exploited in large scale genetic screens or selections. As a result, large plasmid-based libraries of protein genes or pools of mutagenized variants of a given gene may be examined in relatively simple genetic experiments. The strain construction technique is also useful for generating E.coli strains bearing reporters for other types of genetic systems, including repression-based and activation-based systems in which chimeric proteins are used to examine interactions between foreign protein domains.

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Selected References

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  • Vieira J, Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. [PubMed]
  • Casadaban MJ, Chou J, Cohen SN. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. [PMC free article] [PubMed]
  • Derman AI, Puziss JW, Bassford PJ, Jr, Beckwith J. A signal sequence is not required for protein export in prlA mutants of Escherichia coli. EMBO J. 1993 Mar;12(3):879–888. [PubMed]
  • Whipple FW, Kuldell NH, Cheatham LA, Hochschild A. Specificity determinants for the interaction of lambda repressor and P22 repressor dimers. Genes Dev. 1994 May 15;8(10):1212–1223. [PubMed]
  • Backman K, Ptashne M. Maximizing gene expression on a plasmid using recombination in vitro. Cell. 1978 Jan;13(1):65–71. [PubMed]
  • Santiago-Rivera ZI, Williams JS, Gorenstein DG, Andrisani OM. Bacterial expression and characterization of the CREB bZip module: circular dichroism and 2D 1H-NMR studies. Protein Sci. 1993 Sep;2(9):1461–1471. [PubMed]
  • Nelson HC, Sauer RT. Lambda repressor mutations that increase the affinity and specificity of operator binding. Cell. 1985 Sep;42(2):549–558. [PubMed]
  • Whipple FW, Ptashne M, Hochschild A. The activation defect of a lambda cI positive control mutant. J Mol Biol. 1997 Jan 24;265(3):261–265. [PubMed]
  • Lee KA, Masson N. Transcriptional regulation by CREB and its relatives. Biochim Biophys Acta. 1993 Sep 23;1174(3):221–233. [PubMed]
  • Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. [PubMed]
  • Harley CB, Reynolds RP. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. [PMC free article] [PubMed]
  • Hu JC, O'Shea EK, Kim PS, Sauer RT. Sequence requirements for coiled-coils: analysis with lambda repressor-GCN4 leucine zipper fusions. Science. 1990 Dec 7;250(4986):1400–1403. [PubMed]
  • Marchetti A, Abril-Marti M, Illi B, Cesareni G, Nasi S. Analysis of the Myc and Max interaction specificity with lambda repressor-HLH domain fusions. J Mol Biol. 1995 May 5;248(3):541–550. [PubMed]
  • Jappelli R, Brenner S. Interaction between cAMP-dependent protein kinase catalytic subunit and peptide inhibitors analyzed with lambda repressor fusions. J Mol Biol. 1996 Jun 21;259(4):575–578. [PubMed]
  • Zeng X, Hu JC. Detection of tetramerization domains in vivo by cooperative DNA binding to tandem lambda operator sites. Gene. 1997 Feb 7;185(2):245–249. [PubMed]
  • Dove SL, Joung JK, Hochschild A. Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature. 1997 Apr 10;386(6625):627–630. [PubMed]
  • Bunker CA, Kingston RE. Identification of a cDNA for SSRP1, an HMG-box protein, by interaction with the c-Myc oncoprotein in a novel bacterial expression screen. Nucleic Acids Res. 1995 Jan 25;23(2):269–276. [PMC free article] [PubMed]
  • Castagnoli L, Vetriani C, Cesareni G. Linking an easily detectable phenotype to the folding of a common structural motif. Selection of rare turn mutations that prevent the folding of Rop. J Mol Biol. 1994 Apr 8;237(4):378–387. [PubMed]
  • Cairns MT, Green AJ, White PM, Johnston PG, Brenner S. A novel bacterial vector system for monitoring protein-protein interactions in the cAMP-dependent protein kinase complex. Gene. 1997 Jan 31;185(1):5–9. [PubMed]
  • Elledge SJ, Sugiono P, Guarente L, Davis RW. Genetic selection for genes encoding sequence-specific DNA-binding proteins. Proc Natl Acad Sci U S A. 1989 May;86(10):3689–3693. [PubMed]

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