Abstract
Mutations in the araC gene of Escherichia coli B/r were isolated which alter both activation of the araBAD operon expression and autoregulation. The mutations were isolated on an araC-containing plasmid by hydroxylamine mutagenesis of plasmid DNA. The mutant phenotype selected was the inability to autoregulate. The DNA sequence of 16 mutants was determined and found to consist of seven different missense mutations located within the distal third of the araC gene. Enzyme activities revealed that each araC mutation had altered both autoregulatory and activator functions of AraC protein. The mutational analysis presented in this paper suggests that both autoregulatory and activator functions are localized to the same determinants of the AraC protein and that the amino acid sequence within the carboxy-terminal region of AraC protein is important for site-specific DNA binding.
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- Bachmann B. J. Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev. 1983 Jun;47(2):180–230. doi: 10.1128/mr.47.2.180-230.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brunelle A., Hendrickson W., Schleif R. Altered DNA contacts made by a mutant AraC protein. Nucleic Acids Res. 1985 Jul 25;13(14):5019–5026. doi: 10.1093/nar/13.14.5019. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casadaban M. J., Chou J., Cohen S. N. 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. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casadaban M. J., Cohen S. N. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4530–4533. doi: 10.1073/pnas.76.9.4530. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casadaban M. J. Regulation of the regulatory gene for the arabinose pathway, araC. J Mol Biol. 1976 Jul 5;104(3):557–566. doi: 10.1016/0022-2836(76)90120-0. [DOI] [PubMed] [Google Scholar]
- Cass L. G., Horwitz A. H., Miyada C. G., Greenfield L., Wilcox G. The araC regulatory gene mRNA contains a leader sequence. Mol Gen Genet. 1980;180(1):219–226. doi: 10.1007/BF00267373. [DOI] [PubMed] [Google Scholar]
- Clarke P., Lin H. C., Wilcox G. The nucleotide sequence of the araC regulatory gene in Salmonella typhimurium LT2. Gene. 1982 May;18(2):157–163. doi: 10.1016/0378-1119(82)90113-5. [DOI] [PubMed] [Google Scholar]
- Covarrubias L., Cervantes L., Covarrubias A., Soberón X., Vichido I., Blanco A., Kupersztoch-Portnoy Y. M., Bolivar F. Construction and characterization of new cloning vehicles. V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328. Gene. 1981 Jan-Feb;13(1):25–35. doi: 10.1016/0378-1119(81)90040-8. [DOI] [PubMed] [Google Scholar]
- Duncan B. K., Rockstroh P. A., Warner H. R. Escherichia coli K-12 mutants deficient in uracil-DNA glycosylase. J Bacteriol. 1978 Jun;134(3):1039–1045. doi: 10.1128/jb.134.3.1039-1045.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dunn T. M., Hahn S., Ogden S., Schleif R. F. An operator at -280 base pairs that is required for repression of araBAD operon promoter: addition of DNA helical turns between the operator and promoter cyclically hinders repression. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5017–5020. doi: 10.1073/pnas.81.16.5017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Englesberg E., Sheppard D., Squires C., Meronk F., Jr An analysis of "revertants" of a deletion mutant in the C gene of the L-arabinose gene complex in Escherichia coli B-r: isolation of initiator constitutive mutants (Ic). J Mol Biol. 1969 Jul 28;43(2):281–298. doi: 10.1016/0022-2836(69)90268-x. [DOI] [PubMed] [Google Scholar]
- Englesberg E., Wilcox G. Regulation: positive control. Annu Rev Genet. 1974;8:219–242. doi: 10.1146/annurev.ge.08.120174.001251. [DOI] [PubMed] [Google Scholar]
- Gielow L., Largen M., Englesberg E. Initiator constitutive mutants of the L-arabinose operon (OIBAD) of Escherichia coli B/r. Genetics. 1971 Nov;69(3):289–302. doi: 10.1093/genetics/69.3.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Greenfield L., Boone T., Wilcox G. DNA sequence of the araBAD promoter in Escherichia coli B/r. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4724–4728. doi: 10.1073/pnas.75.10.4724. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hahn S., Schleif R. In vivo regulation of the Escherichia coli araC promoter. J Bacteriol. 1983 Aug;155(2):593–600. doi: 10.1128/jb.155.2.593-600.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hendrickson W., Schleif R. F. Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay. J Mol Biol. 1984 Sep 25;178(3):611–628. doi: 10.1016/0022-2836(84)90241-9. [DOI] [PubMed] [Google Scholar]
- Hendrickson W., Schleif R. A dimer of AraC protein contacts three adjacent major groove regions of the araI DNA site. Proc Natl Acad Sci U S A. 1985 May;82(10):3129–3133. doi: 10.1073/pnas.82.10.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horwitz A. H., Heffernan L., Morandi C., Lee J. H., Timko J., Wilcox G. DNA sequence of the araBAD-araC controlling region in Salmonella typhimurium LT2. Gene. 1981 Sep;14(4):309–319. doi: 10.1016/0378-1119(81)90163-3. [DOI] [PubMed] [Google Scholar]
- Horwitz A. H., Miyada C. G., Wilcox G. Functional limits of the araIc promoter suggest an additional regulatory site for araBAD expression. J Bacteriol. 1984 Apr;158(1):141–147. doi: 10.1128/jb.158.1.141-147.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Isackson P. J., Bertrand K. P. Dominant negative mutations in the Tn10 tet repressor: evidence for use of the conserved helix-turn-helix motif in DNA binding. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6226–6230. doi: 10.1073/pnas.82.18.6226. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ito H., Ike Y., Ikuta S., Itakura K. Solid phase synthesis of polynucleotides. VI. Further studies on polystyrene copolymers for the solid support. Nucleic Acids Res. 1982 Mar 11;10(5):1755–1769. doi: 10.1093/nar/10.5.1755. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kelley R. L., Yanofsky C. Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA. Proc Natl Acad Sci U S A. 1985 Jan;82(2):483–487. doi: 10.1073/pnas.82.2.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee N. L., Gielow W. O., Wallace R. G. Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli. Proc Natl Acad Sci U S A. 1981 Feb;78(2):752–756. doi: 10.1073/pnas.78.2.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lei S. P., Lin H. C., Heffernan L., Wilcox G. araB Gene and nucleotide sequence of the araC gene of Erwinia carotovora. J Bacteriol. 1985 Nov;164(2):717–722. doi: 10.1128/jb.164.2.717-722.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McKay D. B., Weber I. T., Steitz T. A. Structure of catabolite gene activator protein at 2.9-A resolution. Incorporation of amino acid sequence and interactions with cyclic AMP. J Biol Chem. 1982 Aug 25;257(16):9518–9524. [PubMed] [Google Scholar]
- Miyada C. G., Horwitz A. H., Cass L. G., Timko J., Wilcox G. DNA sequence of the araC regulatory gene from Escherichia coli B/r. Nucleic Acids Res. 1980 Nov 25;8(22):5267–5274. doi: 10.1093/nar/8.22.5267. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miyada C. G., Stoltzfus L., Wilcox G. Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4120–4124. doi: 10.1073/pnas.81.13.4120. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Novick R. P., Clowes R. C., Cohen S. N., Curtiss R., 3rd, Datta N., Falkow S. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol Rev. 1976 Mar;40(1):168–189. doi: 10.1128/br.40.1.168-189.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ogden S., Haggerty D., Stoner C. M., Kolodrubetz D., Schleif R. The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3346–3350. doi: 10.1073/pnas.77.6.3346. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith B. R., Schleif R. Nucleotide sequence of the L-arabinose regulatory region of Escherichia coli K12. J Biol Chem. 1978 Oct 10;253(19):6931–6933. [PubMed] [Google Scholar]
- Steitz T. A., Ohlendorf D. H., McKay D. B., Anderson W. F., Matthews B. W. Structural similarity in the DNA-binding domains of catabolite gene activator and cro repressor proteins. Proc Natl Acad Sci U S A. 1982 May;79(10):3097–3100. doi: 10.1073/pnas.79.10.3097. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sutcliffe J. G. pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long. Nucleic Acids Res. 1978 Aug;5(8):2721–2728. doi: 10.1093/nar/5.8.2721. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tweten R. K., Collier R. J. Molecular cloning and expression of gene fragments from corynebacteriophage beta encoding enzymatically active peptides of diphtheria toxin. J Bacteriol. 1983 Nov;156(2):680–685. doi: 10.1128/jb.156.2.680-685.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wallace R. B., Johnson M. J., Suggs S. V., Miyoshi K., Bhatt R., Itakura K. A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322. Gene. 1981 Dec;16(1-3):21–26. doi: 10.1016/0378-1119(81)90057-3. [DOI] [PubMed] [Google Scholar]
- Wallace R. G., Lee N., Fowler A. V. The araC gene of Escherichia coli: transcriptional and translational start-points and complete nucleotide sequence. Gene. 1980 Dec;12(3-4):179–190. doi: 10.1016/0378-1119(80)90100-6. [DOI] [PubMed] [Google Scholar]
- Weber I. T., Steitz T. A. Model of specific complex between catabolite gene activator protein and B-DNA suggested by electrostatic complementarity. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3973–3977. doi: 10.1073/pnas.81.13.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilcox G., Boulter J., Lee N. Direction of transcription of the regulatory gene araC in Escherichia coli B-r. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3635–3639. doi: 10.1073/pnas.71.9.3635. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilcox G., Clemetson K. J., Santi D. V., Englesberg E. Purification of the araC protein. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2145–2148. doi: 10.1073/pnas.68.9.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilcox G., Meuris P. Stabilization and size of araC protein. Mol Gen Genet. 1976 Apr 23;145(1):97–100. doi: 10.1007/BF00331563. [DOI] [PubMed] [Google Scholar]