Evidence that a single monomer of Spx can productively interact with RNA polymerase in Bacillus subtilis

Spx activates transcription initiation in Bacillus subtilis by directly interacting with the C-terminal domain of the RNA polymerase(RNAP) holoenzyme- subunit, which generates a complex that recognizes the promoter regions of genes within the Spxregulon. Many Gram-positive species possess multiple paralogs of Spx, suggesting that two paralogous forms of Spx could simultaneouslycontact RNAP. The composition of Spx/RNAP was examined in vitro using an Spx variant (SpxδCHA) bearing a 12-amino-acid deletion of the C terminus (SpxδC) and a hemagglutinin (HA) epitope tag and Spxc-Myc, a full-length Spx with aC-terminal myelocytomatosis oncoprotein (c-Myc) epitope tag. All Spx/RNAP complexes bearing deletion or C-terminal-taggedvariants were transcriptionally active in vivo and in vitro. Reaction mixtures containing SpxδCHA and Spxc-Myc combinedwith RNAP were applied to either anti-HA or anti-c-Myc affinity columns. Eluted fractions contained RNAP with only one of theepitope-tagged Spx derivatives. The resin-bound RNAP complex bearing a single epitope-tagged Spx derivative was transcriptionallyactive. In vivo production of SpxδC and SpxδCHA followed by anti-HA affinity column chromatography of a clearedlysate resulted in retrieval of Spx/RNAP with only the SpxδCHA derivative. Binding reactions that combined active Spxc-Myc, inactive Spx(R60E)δCHA, and RNAP, when applied to the anti-HA affinity column, yielded only inactive Spx(R60E)δCHA/RNAP complexes. The results strongly argue for a model in which a single Spx monomer engages RNAP to generate an activetranscriptional complex.