Regulation of arginine biosynthesis in the psychropiezophilic bacterium Moritella profunda: in vivo repressibility and in vitro repressor-operator contact probing
| Autor: | Daniel Charlier, Nicolas Glansdorff, Ying Xu, Yuan Y. Sun, Daniel Gigot, Nadine Huysveld |
|---|---|
| Přispěvatelé: | Microbiology, Department of Bio-engineering Sciences, Vrije Universiteit Brussel |
| Rok vydání: | 2003 |
| Předmět: |
DNA
Bacterial Hot Temperature Operator Regions Genetic Transcription Genetic Operon Protein subunit Mutant Molecular Sequence Data DNA Footprinting Repressor In Vitro Techniques Regulatory Sequences Nucleic Acid Arginine Amidohydrolases Bacterial Proteins Structural Biology Consensus Sequence Escherichia coli Amino Acid Sequence Cloning Molecular Promoter Regions Genetic Molecular Biology Peptide sequence Genetics biology Base Sequence Sequence Homology Amino Acid Escherichia coli Proteins Promoter Gene Expression Regulation Bacterial biology.organism_classification Aldehyde Oxidoreductases Footprinting Recombinant Proteins Repressor Proteins Thermotoga maritima Mutagenesis Site-Directed Nucleic Acid Conformation Moritella Protein Binding |
| Zdroj: | Vrije Universiteit Brussel |
| ISSN: | 0022-2836 |
| Popis: | We report the cloning of the arginine repressor gene from the psychropiezophilic Gram-negative bacterium Moritella profunda , the purification of its product (ArgR Mp ), the identification of the operator in the bipolar argECBFGH ( A ) operon, in vivo repressibility studies, and an in vitro analysis of the repressor–operator interaction, including binding to mutant and heterologous arginine operators. The ArgR Mp subunit shows about 70% amino acid sequence identity with Escherichia coli ArgR (ArgR Ec ). Binding of purified hexameric ArgR Mp to the control region of the divergent operon proved to be arginine-dependent, sequence-specific, and significantly more sensitive to heat than complex formation with ArgR Ec . ArgR Mp binds E. coli arginine operators very efficiently, but hardly recognizes the operator from Bacillus stearothermophilus or Thermotoga maritima . ArgR Mp binds to a single site overlapping the −35 element of argC P , but not argE P . Therefore, the arrangement of promoter and operator sites in the bipolar argECBFGH ( A ) operon of M. profunda is very different from the organization of control elements in the bipolar argECBH operon of E. coli , where both promoters overlap the common operator and are equally repressible. We demonstrate that M. profunda argC P is about 44-fold repressible, whereas argE P is fully constitutive. A high-resolution contact map of the ArgR Mp –operator interaction was established by enzymatic and chemical footprinting, missing contact and base-specific premodification binding interference studies. The results indicate that the argC operator consists of two ARG box-like sequences (18 bp imperfect palindromes) separated by 3 bp. ArgR Mp binds to one face of the DNA helix and establishes contacts with two major groove segments and the intervening minor groove of each ARG box, whereas the minor groove segment facing the repressor at the center of the operator remains largely uncontacted. This pattern is reminiscent of complex formation with the repressors of E. coli and B. stearothermophilus , and suggests that each ARG box is contacted by two ArgR subunits belonging to opposite trimers. Moreover, the premodification interference patterns and mutant studies clearly indicate that the inner, center proximal halves of each ARG box in the M. profunda argC operator are more important for complex formation and repression than the outermost halves. A close inspection of sequence conservation and of single base-pair O c -type mutations indicate that the same conclusion can be generalized to E. coli operators. |
| Databáze: | OpenAIRE |
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