title: Protein crosslinking studies suggest that Rhizobium meliloti C4-dicarboxylic acid transport protein D, a sigma 54-dependent transcriptional activator, interacts with sigma 54 and the beta subunit of RNA polymerase.
abstract: Rhizobium meliloti C4-dicarboxylic acid transport protein D (DCTD) activates transcription by a form of RNA polymerase holoenzyme that has sigma 54 as its sigma factor (referred to as E sigma 54). DCTD catalyzes the ATP-dependent isomerization of closed complexes between E sigma 54 and the dctA promoter to transcriptionally productive open complexes. Transcriptional activation probably involves specific protein-protein interactions between DCTD and E sigma 54. Interactions between sigma 54-dependent activators and E sigma 54 are transient, and there has been no report of a biochemical assay for contact between E sigma 54 and any activator to date. Heterobifunctional crosslinking reagents were used to examine protein-protein interactions between the various subunits of E sigma 54 and DCTD. DCTD was crosslinked to Salmonella typhimurium sigma 54 with the crosslinking reagents succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-hydroxysulfosuccinimidyl-4-azidobenzoate. Cys-307 of sigma 54 was identified by site-directed mutagenesis as the residue that was crosslinked to DCTD. DCTD was also crosslinked to the beta subunit of Escherichia coli core RNA polymerase with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, but not with N-hydroxysulfosuccinimidyl-4-azidobenzoate. These data suggest that interactions of DCTD with sigma 54 and the beta subunit may be important for transcriptional activation and offer evidence for interactions between a sigma 54-dependent activator and sigma 54, as well as the beta subunit of RNA polymerase.
authors.affiliation: Department of Microbiology, University of Georgia, Athens 30602, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Lee
authors.forename: T R
authors.initials: TR
authors.lastname: Hoover
mesh_terms: D001386:Azides; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D003998:Dicarboxylic Acids; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D008301:Maleimides; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54; D012486:Salmonella typhimurium / Q000235:genetics; D017384:Sequence Deletion; D012808:Sigma Factor / Q000737:chemistry / Q000378:metabolism*; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001386:Azides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D003998:Dicarboxylic Acids; D029968:Escherichia coli Proteins; D008301:Maleimides; C096033:N-hydroxysulfosuccinimidyl-4-azidobenzoate; D012808:Sigma Factor; D014157:Transcription Factors; C497751:rpoN protein, E coli; C071675:sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; C073788:dctD protein, Rhizobium meliloti; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54