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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 92; no. 21; pp. 9702 - 9706
• Main Authors: Lee, J.H. (University of Georgia, Athens, GA.), Hoover, T.R
• Format: Journal Article
• Language: English
• Published: United States National Acad Sciences 10.10.1995; National Academy of Sciences
• Subjects: Azides; BACTERIA; Bacterial Proteins - metabolism; Base Sequence; Biochemistry; Biological Transport; Cross-Linking Reagents; Dicarboxylic Acids; DNA-Binding Proteins; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - metabolism; Escherichia coli - enzymology; Escherichia coli Proteins; GENETICA; GENETIQUE; ISOMERISATION; ISOMERIZACION; Maleimides; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding; PROTEINAS; PROTEINAS AGLUTINANTES; PROTEINE; PROTEINE DE LIAISON; Proteins; RHIZOBIUM MELILOTI; Ribonucleic acid; RNA; RNA Polymerase Sigma 54; Salmonella typhimurium - genetics; Sequence Deletion; Sigma Factor - chemistry; Sigma Factor - metabolism; Sinorhizobium meliloti - genetics; Sinorhizobium meliloti - metabolism; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - metabolism; TRANSFERASAS; TRANSFERASE
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.21.9702

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)cyelohexane-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