Summary
The product of the Klebsiella pneumoniae nitrogen regulatory gene ntrC has been purified and shown to be a dimeric protein of subunit molecular weight 54Kd, designated NtrC. In an in vitro coupled transcription-translation system NtrC inhibited expression from both the ntrBC and glnA promoters. NtrC bound to both of these ntr repressible promoters with equal affinity, but did not bind to the activatable nitrogen fixation promoters nifF of nifLA. NtrC makes contact with nucleotides flanking the — 10 region of the glnA (RNA2) promoter at sequences homologous with the proposed consensus binding site.
Similar content being viewed by others
References
Alvarez-Morales A, Dixon R, Merrick M (1984) Positive and negative control of the glnA ntrBC regulon in Klebsiella pneumoniae. EMBO J 3: 501–507
Ames Ferro-Luzzi G, Nikaido K (1985) Nitrogen regulation in Salmonella typhimurium. Identification of an ntrC protein-binding site and definition of a consensus binding sequence. EMBO J 4: 539–547
Beynon J, Cannon M, Buchanan-Wollaston V, Cannon FC (1983) The nif promoters of Klebsiella pneumoniae have a characteristic primary structure. Cell 34: 665–671
Casadaban MJ, Chou J, Cohen S (1980) In vitro gene fusions that join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the deletion and cloning of translational initiation signals. J Bacteriol 143: 971–980
Close TJ, Rodriguez RL (1982) Construction and characterisation of the chloramphenicol-resistance gene cartridge: A new approach to the transcriptional mapping of extrachromosomal elements. Gene 20: 305–316
Collins J (1979) Cell free synthesis of proteins coding for mobilisation functions of ColE1. Gene 6: 29–42
Dixon R (1984) Tandem promoters determine regulation of the Klebsiella pneumoniae glutamine synthetase (glnA) gene. Nucleic Acids Res 12: 7811–7830
Dixon R, Alvarez-Morales A, Clements J, Drummond M, Filser M, Merrick M (1983) Regulation of transcription of the nitrogen fixation operons. In: Voellmy R, Downey K, Ahmed F, Schultz J (eds) Molecular genetics of plants and animals, Miami Winter Symposium, vol 20, Academic Press, New York, p 223
Drummond M, Clements J, Merrick M, Dixon R (1983) Positive control and autogenous regulation of the nifLA promoter in Klebsiella pneumoniae. Nature 301: 302–307
Espin G, Alvarez-Morales A, Cannon F, Dixon R, Merrick M (1982) Cloning of the glnA, ntrB and ntrC genes of Klebsiella pneumoniae and studies of their role in regulation of the nitrogen fixation (nif) gene cluster. Mol Gen Genet 185: 518–524
Fried M, Crothers D (1981) Equilibria and kinetics of lac repressoroperator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res 9: 6505–6525
Galas D, Schmitz A (1978) DNase footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res 5: 3157–3170
Garner MM, Revzin A (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res 9: 3047–3060
Itzaki RF, Gill DM (1964) A micro-biuret method for estimating proteins. Anal Biochem 9: 401–410
Kröger M, Hobom G (1982) Structural analysis of insertion sequence IS5. Nature 297: 159–162
MacNeil T, Roberts GP, MacNeil D, Tyler B (1982) The products of glnL and glnG are bifunctional regulatory proteins. Mol Gen Genet 188: 325–333
McFarland N, McCarter L, Artz S, Kustu S (1981) Nitrogen regulatory locus (glnR) of enteric bacteria is composed of cistrons ntrB and ntrC: identification of their protein products. Proc Natl Acad Sci USA 78: 2135–2139
Magasanik B (1982) Genetic control of nitrogen assimilation in bacteria. Annu Rev Genet 16: 135–168
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Maxam A, Gilbert W (1980) Sequencing end-labelled DNA with base-specific chemical cleavages. Meth Enzymol 65: 499–559
Merrick M (1983) Nitrogen control of the nif regulon in Klebsiella pneumoniae: involvement of the ntrA gene and analogies between ntrC and nifA. EMBO J 2: 39–44
Ow DW, Ausubel FM (1983) Regulation of nitrogen metabolism genes by the nifA gene product. Nature 301: 307–313
Ow DW, Sundaresan V, Rothstein DM, Brown SE, Ausubel F (1983) Promoters regulated by the glnG (ntrC) and nifA gene products share a heptameric consensus sequence in the — 15 region. Proc Natl Acad Sci USA 80: 2524–2528
Pabo CO, Sauer RT (1984) Protein-DNA Recognition. Annu Rev Biochem 53: 293–321
Reitzer LJ, Magasanik B (1983) Isolation of the nitrogen assimilation regulator NRI the product of the glnG gene of Escherichia coli. Proc Natl Acad Sci USA 80: 5554–5558
Sollner-Webb B, Reeder RH (1979) The nucleotide sequence of the initiation and termination sites for ribosomal transcription in X. laevis. Cell 18: 485–499
Ueno-Nishio S, Mango S, Reitzer LJ, Magasanik B (1984) Identification and regulation of the glnL promoter of the complex glnALG operon of Escherichia coli. J Bacteriol 160: 379–384
Wei GR, Kustu S (1981) Glutamine auxotrophs with mutations in the nitrogen regulatory gene ntrC that is near glnA. Mol Gen Genet 183: 392–399
Zubay G (1973) In vitro synthesis of protein in microbial systems. Annu Rev Genet 7: 267–287
Author information
Authors and Affiliations
Additional information
Communicated by C. Auerbach
Rights and permissions
About this article
Cite this article
Hawkes, T., Merrick, M. & Dixon, R. Interaction of purified NtrC protein with nitrogen regulated promoters from Klebsiella pneumoniae . Molec Gen Genet 201, 492–498 (1985). https://doi.org/10.1007/BF00331345
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00331345