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. 1992 Oct 15;287(Pt 2):493–499. doi: 10.1042/bj2870493

Overexpression, purification and characterization of the Escherichia coli MelR transcription activator protein.

R Caswell 1, J Williams 1, A Lyddiatt 1, S Busby 1
PMCID: PMC1133192  PMID: 1445207

Abstract

The gene encoding Escherichia coli MelR protein has been cloned in the expression vector pJLA502. MelR has been overexpressed, substantially purified and shown to bind to DNA fragments carrying the melAB promoter. A truncated version of the melR gene, encoding the C-terminal half of MelR, was also cloned into pJLA502; the protein product of this truncated gene binds to the melAB promoter but was not overproduced. A number of amino acid substitutions were made in the recognition helices of two putative helix-turn-helix motifs in the C-terminal part of MelR, and the effects of these mutations on MelR-dependent transcription initiation at the melAB promoter have been measured.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Brunelle A., Schleif R. Determining residue-base interactions between AraC protein and araI DNA. J Mol Biol. 1989 Oct 20;209(4):607–622. doi: 10.1016/0022-2836(89)90598-6. [DOI] [PubMed] [Google Scholar]
  3. Busby S., Dreyfus M. Segment-specific mutagenesis of the regulatory region in the Escherichia coli galactose operon: isolation of mutations reducing the initiation of transcription and translation. Gene. 1983 Jan-Feb;21(1-2):121–131. doi: 10.1016/0378-1119(83)90154-3. [DOI] [PubMed] [Google Scholar]
  4. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  5. Caswell R., Webster C., Busby S. Studies on the binding of the Escherichia coli MelR transcription activator protein to operator sequences at the MelAB promoter. Biochem J. 1992 Oct 15;287(Pt 2):501–508. doi: 10.1042/bj2870501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DiRita V. J. Co-ordinate expression of virulence genes by ToxR in Vibrio cholerae. Mol Microbiol. 1992 Feb;6(4):451–458. doi: 10.1111/j.1365-2958.1992.tb01489.x. [DOI] [PubMed] [Google Scholar]
  7. Francklyn C. S., Lee N. AraC proteins with altered DNA sequence specificity which activate a mutant promoter in Escherichia coli. J Biol Chem. 1988 Mar 25;263(9):4400–4407. [PubMed] [Google Scholar]
  8. Frank D. W., Iglewski B. H. Cloning and sequence analysis of a trans-regulatory locus required for exoenzyme S synthesis in Pseudomonas aeruginosa. J Bacteriol. 1991 Oct;173(20):6460–6468. doi: 10.1128/jb.173.20.6460-6468.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hanatani M., Yazyu H., Shiota-Niiya S., Moriyama Y., Kanazawa H., Futai M., Tsuchiya T. Physical and genetic characterization of the melibiose operon and identification of the gene products in Escherichia coli. J Biol Chem. 1984 Feb 10;259(3):1807–1812. [PubMed] [Google Scholar]
  10. Henikoff S., Wallace J. C., Brown J. P. Finding protein similarities with nucleotide sequence databases. Methods Enzymol. 1990;183:111–132. doi: 10.1016/0076-6879(90)83009-x. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Liljeström P. L., Liljeström P. Nucleotide sequence of the melA gene, coding for alpha-galactosidase in Escherichia coli K-12. Nucleic Acids Res. 1987 Mar 11;15(5):2213–2220. doi: 10.1093/nar/15.5.2213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lobell R. B., Schleif R. F. AraC-DNA looping: orientation and distance-dependent loop breaking by the cyclic AMP receptor protein. J Mol Biol. 1991 Mar 5;218(1):45–54. doi: 10.1016/0022-2836(91)90872-4. [DOI] [PubMed] [Google Scholar]
  14. Lodge J., Williams R., Bell A., Chan B., Busby S. Comparison of promoter activities in Escherichia coli and Pseudomonas aeruginosa: use of a new broad-host-range promoter-probe plasmid. FEMS Microbiol Lett. 1990 Jan 15;55(1-2):221–225. doi: 10.1016/0378-1097(90)90199-z. [DOI] [PubMed] [Google Scholar]
  15. Menon K. P., Lee N. L. Activation of ara operons by a truncated AraC protein does not require inducer. Proc Natl Acad Sci U S A. 1990 May;87(10):3708–3712. doi: 10.1073/pnas.87.10.3708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ramos J. L., Rojo F., Zhou L., Timmis K. N. A family of positive regulators related to the Pseudomonas putida TOL plasmid XylS and the Escherichia coli AraC activators. Nucleic Acids Res. 1990 Apr 25;18(8):2149–2152. doi: 10.1093/nar/18.8.2149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schauder B., Blöcker H., Frank R., McCarthy J. E. Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region. Gene. 1987;52(2-3):279–283. doi: 10.1016/0378-1119(87)90054-0. [DOI] [PubMed] [Google Scholar]
  18. Shimamoto T., Yazyu H., Futai M., Tsuchiya T. Nucleotide sequence of the promoter region of the melibiose operon of Escherichia coli. Biochem Biophys Res Commun. 1984 May 31;121(1):41–46. doi: 10.1016/0006-291x(84)90685-5. [DOI] [PubMed] [Google Scholar]
  19. Steitz T. A. Structural studies of protein-nucleic acid interaction: the sources of sequence-specific binding. Q Rev Biophys. 1990 Aug;23(3):205–280. doi: 10.1017/s0033583500005552. [DOI] [PubMed] [Google Scholar]
  20. Tobin J. F., Schleif R. F. Purification and properties of RhaR, the positive regulator of the L-rhamnose operons of Escherichia coli. J Mol Biol. 1990 Jan 5;211(1):75–89. doi: 10.1016/0022-2836(90)90012-B. [DOI] [PubMed] [Google Scholar]
  21. 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. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  22. Webster C., Gardner L., Busby S. The Escherichia coli melR gene encodes a DNA-binding protein with affinity for specific sequences located in the melibiose-operon regulatory region. Gene. 1989 Nov 30;83(2):207–213. doi: 10.1016/0378-1119(89)90106-6. [DOI] [PubMed] [Google Scholar]
  23. Webster C., Gaston K., Busby S. Transcription from the Escherichia coli melR promoter is dependent on the cyclic AMP receptor protein. Gene. 1988 Sep 7;68(2):297–305. doi: 10.1016/0378-1119(88)90032-7. [DOI] [PubMed] [Google Scholar]
  24. Webster C., Kempsell K., Booth I., Busby S. Organisation of the regulatory region of the Escherichia coli melibiose operon. Gene. 1987;59(2-3):253–263. doi: 10.1016/0378-1119(87)90333-7. [DOI] [PubMed] [Google Scholar]

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