ABSTRACT
The bgl sensory system is composed of a membrane-bound sugar sensor, BglF, and a transcriptional regulator, BglG. The sensor BglF has several enzymatic activities: in its nonstimulated state, it acts as BglG phosphorylase; in the presence of beta-glucoside in the growth medium, it acts as BglG dephosphorylase and as the beta-glucoside phosphotransferase. The same active site on BglF, Cys-24, is responsible for the phosphorylation of both the stimulating sugar and the BglG protein. BglF is composed of three domains, two hydrophilic and one hydrophobic. Our previous results suggested that catalysis of the sugar-stimulated functions depends on specific interactions between the B domain, which contains the active site cysteine, and the integral membrane C domain. We report here that the stimulating sugar triggers the formation of a disulfide bond between the active site cysteine and another cysteine in the membrane-embedded domain of BglF. Inability of a mutant BglF protein to form the disulfide bridge between the B and C domains correlates with its inability to catalyze the sugar-stimulated functions. The ability of the cysteine residue in BglF to bind covalently either to a phosphoryl group or to another cysteine residue, depending on the protein stimulation state, suggests a novel way to control signaling by alternative bond formation.
Subject(s)
Bacterial Proteins/metabolism , Carbohydrate Metabolism , Escherichia coli Proteins , Membrane Proteins/physiology , Protein Kinases/physiology , RNA-Binding Proteins/metabolism , Bacterial Proteins/genetics , Binding Sites , Cysteine/chemistry , Cysteine/metabolism , Disulfides , Electrophoresis, Polyacrylamide Gel , Glucosides/metabolism , Membrane Proteins/chemistry , Models, Biological , Phosphorylation , Plasmids/metabolism , Protein Binding , Protein Kinases/chemistry , Protein Structure, Tertiary , RNA-Binding Proteins/genetics , Signal Transduction , Time Factors , Transcription, GeneticABSTRACT
The Escherichia coli BglG protein antiterminates transcription at two terminator sites within the bgl operon in response to the presence of beta-glucosides in the growth medium. BglG was previously shown to be an RNA-binding protein that recognizes a specific sequence located just upstream of each of the terminators and partially overlapping with them. We show here that BglG also binds to the E. coli RNA polymerase, both in vivo and in vitro. By using several techniques, we identified the beta' subunit of RNA polymerase as the target for BglG binding. The region that contains the binding site for BglG was mapped to the N-terminal region of beta'. The beta' subunit, produced in excess, prevented BglG activity as a transcriptional antiterminator. Possible roles of the interaction between BglG and the polymerase beta' subunit are discussed.
Subject(s)
ATP-Binding Cassette Transporters , Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/metabolism , Escherichia coli Proteins , Escherichia coli/metabolism , Monosaccharide Transport Proteins , RNA-Binding Proteins/metabolism , Bacterial Proteins/isolation & purification , Blotting, Western , Carrier Proteins/biosynthesis , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Chromatography, Affinity , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/isolation & purification , Macromolecular Substances , Maltose-Binding Proteins , Plasmids , RNA-Binding Proteins/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sequence DeletionABSTRACT
The Escherichia coli transcriptional antiterminator protein BglG inhibits transcription termination of the bgl operon in response to the presence of beta-glucosides in the growth medium. BglG is an RNA-binding protein that recognizes a specific sequence partially overlapping the two terminators within the bgl transcript. The activity of BglG is determined by its dimeric state which is modulated by reversible phosphorylation. Thus, only the nonphosphorylated dimer binds to the RNA target site and allows readthrough of transcription. Genetic systems which test dimerization and antitermination in vivo were used to map and delimit the region which mediates BglG dimerization. We show that the last 104 residues of BglG are required for dimerization. Any attempt to shorten this region from the ends or to introduce internal deletions abolished the dimerization capacity of this region. A putative leucine zipper motif is located at the N terminus of this region. The role of the canonical leucines in dimerization was demonstrated by their substitution. Our results also suggest that the carboxy-terminal 70 residues, which follow the leucine zipper, contain another dimerization domain which does not resemble any known dimerization motif. Each of these two regions is necessary but not sufficient for dimerization. The BglG phosphorylation site, His208, resides at the junction of the two putative dimerization domains. Possible mechanisms by which the phosphorylation of BglG controls its dimerization and thus its activity are discussed.
