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 α subunit, which generates a complex that recognizes the promoter regions of genes within the Spx regulon. Many Gram-positive species possess multiple paralogs of Spx, suggesting that two paralogous forms of Spx could simultaneously contact 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 a C-terminal myelocytomatosis oncoprotein (c-Myc) epitope tag. All Spx/RNAP complexes bearing deletion or C-terminal-tagged variants were transcriptionally active in vivo and in vitro. Reaction mixtures containing SpxΔCHA and Spxc-Myc combined with RNAP were applied to either anti-HA or anti-c-Myc affinity columns. Eluted fractions contained RNAP with only one of the epitope-tagged Spx derivatives. The resin-bound RNAP complex bearing a single epitope-tagged Spx derivative was transcriptionally active. In vivo production of SpxΔC and SpxΔCHA followed by anti-HA affinity column chromatography of a cleared lysate 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 active transcriptional complex.
Division of Environmental & Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Beaverton, OR, USA.
Ann A
AA
Lin
Peter
P
Zuber
D020816:Amino Acid Motifs; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D012321:DNA-Directed RNA Polymerases