PROTEIN CROSS-LINKING STUDIES SUGGEST THAT RHIZOBIUM-MELILOTI C-4-DICARBOXYLIC ACID TRANSPORT PROTEIN-D, A SIGMA(54)-DEPENDENT TRANSCRIPTIONAL ACTIVATOR, INTERACTS WITH SIGMA(54)-SUBUNIT AND THE BETA-SUBUNIT OF RNA-POLYMERASE

Rhizobium meliloti C-4-dicarboxylic acid transport protein D (DCTD) activates transcription by a form of RNA polymerase holoenzyme that has sigma(54) as its a 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-l-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.