The activity of ¢B, a secondary cf factor of Bacillus subtilis, is primarily controlled by an anti-af factor protein (RsbW) that binds to ofB and blocks its ability to form an RNA polymerase holoenzyme (E-UrB). Inhibition of ifB by RsbW is counteracted by RsbV, a protein that is essential for the activation of orB-dependent transcription. When crude B. subtilis extracts were fractionated by gel filtration chromatography or electrophoresis through nondenaturing polyacrylamide gels, a complex composed of RsbW and RsbV that is distinct from the previously observed RsbW-CiB complex was detected. In analogous experiments, RsbX, an additional regulator of crB-dependent transcription that is thought to act independently of RsbV-RsbW, was not found to associate with any of the other sigB operon products. Two forms of RsbV were visualized when crude cell extracts of B. subtilis were subjected to isoelectric focusing (IEF), with the more negatively charged RsbV species absent from extracts prepared from RsbW-strains. In vitro, RsbV became phosphorylated when incubated with ATP and RsbW but not with ATP alone. The phosphorylated RsbV species comigrated during IEF with the RsbW-dependent form of RsbV found in crude cell extracts. These results suggest that the modified RsbV, present in crude cell extracts, is phosphorylated. When gel filtration fractions containing RsbV-RsbW complexes or RsbV alone were subjected to IEF, only the unmodified form of RsbV was found associated with RsbW. The presumed phosphorylated variant of RsbV was present only in fractions that did not contain RsbW. The data support a model whereby RsbV binds directly to RsbW and blocks its ability to form the RsbW-crB complex. This activity of RsbV appears to be inhibited by RsbW-dependent phosphorylation.uB is a secondary a factor of Bacillus subtilis. RNA polymerase containing uB (E-aB) can be isolated from vegetatively growing and stationary-phase cells but not from cells that are undergoing sporulation (14-16). Null mutations in the uB structural gene (sigB) confer no obvious phenotype on strains which carry them (6, 13); however, genes which depend on aB for their expression are induced following heat shock or entry of B. subtilis into stationary phase (4,7,9,17,25). This pattern of uB-dependent gene expression suggests that aB plays a role, albeit a nonessential one, in the adaption of B. subtilis to certain environmental stresses.sigB is the third gene of a four-gene operon that is transcribed from a uB-dependent promoter (13,19). The operon's remaining three genes (rsbV, rsbW, and rsbX) have been shown in genetic and biochemical studies to encode regulators of cB (2,3,5,8,18). Null mutations in rsbW or rsbX result in dramatic increases in the expression of uB-dependent gene expression, while the loss of RsbV lowers this expression to levels comparable to that seen in mutant B. subtilis strains that lack aB itself (2, 8, 18). The requirement for RsbV in the expression of aB-dependent genes can be bypassed by mutations in rsbW but not rsbX (2, 8). T...

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A modified form of Bacillus subtifis RNA polymerase (RNA nucleotidyltransferase) has been isolated that exhibits distinctive transcriptional specificity. This modified enzyme transcribes two cloned genes from the purA-cysA region of the B. subtilis chromosome whose expression in vivo is associated with the process of sporulation. Neither of these genes is transcribed by tie usual form of B. subtilis RNA polymerase holoenzyme containing a a factor of 55,000 daltons (I4 The modified RNA polymerase lacks -55 but contains a newly identified subunit of 37,000 daltons termed oP7. A reconstitution experiment in which (P was added to core RNA polymerase strongly suggests that (37 is responsible for the transcriptional specificity of the modified RNA polymerase. (37 apparently acts at the level of promoter recognition; this transcriptional determinant enabled core RNA polymerase to form stable binary and ternary ("initiation") complexes with endonuclease restriction fragments containing promoters for the cloned B. subtilis genes. The first step in transcription of genes in bacteria is the formation of a binary complex of RNA polymerase (RNA nucleotidyltransferase) with promoter sites on DNA (1). The recognition of promoters is catalyzed by a subunit of RNA polymerase known as a. This polypeptide confers on the core component (f'(3a2) of the bacterial transcriptase the ability to bind at and initiate RNA synthesis from specific sites on the DNA template. The discovery that a factor governs site selection in bacteria prompted the proposal that different species of a polypeptide could recognize different classes of promoters (2). Indeed, certain bacteriophage are now known to encode a-like proteins that direct the transcription of phage genes. In Bacillus subtilis phage SPO1 (and its close relative SP82), phage-coded a-like factors replace the host a factor on the B. subtilis transcriptase, thereby converting the bacterial RNA polymerase to a form that recognizes phage "middle" (3-6) and "late" gene promoters (7,8). The promoters for phage "middle" genes have distinctive nucleotide sequences that differ strikingly from the promoters controlled by the host a factor (9).Until now multiple species of a factor in uninfected bacteria have not been described. Recently, however, we (10) isolated a modified form of B. subtilis RNA polymerase that transcribes two cloned B. subtilis genes whose transcription in vivo is associated with the process of sporulation (11-13). Neither of these genes is transcribed by the usual form of B. subtilis RNA polymerase holoenzyme (14, 15) containing a a factor of 55,000 daltons (a55). The modified RNA polymerase lacks a'5 but contains a newly identified subunit of 37,000 daltons. Here we show by means of a reconstitution experiment that the 37,000-dalton polypeptide (herein termed o37) is a a-like factor that confers novel promoter recognition properties on core RNA polymerase. Thus, B. subtilis core RNA polymerase interacts with a variety of bacterial and phage-coded a factors to acquire ...

oB is a secondary ofactor ofBaciUus subtilis.RNA polymerase containing cr transcribes a subset of genes that are expressed after heat shock or the onset of the stationary phase of growth. Three genes (rsbV, rsbW, and rsbX), cotranscribed with the oB structural gene (sigB), regulate o dependent gene expression. RsbW is the primary inhibitor of this system with the other gene products acting upstream of RsbW in the oB regulatory pathway. Evidence is now presented that RsbW inhibits oB-dependent transcription by binding to crB and blocking the formation of a oB-containing RNA polymerase holoenzyme. Antibodies specific for either RsbW or oc will coprecipitate both proteins from crude ceil extracts. This is not due to the presence of both proteins on RNA polymerase. Western blot analysis of B. subtilis extracts that had been fractionated by gel-fitration chromatography revealed a single peak of RsbW that did not coelute with RNA polymerase and two peaks of oB protein: one that eluted with RNA polymerase and a second that overlapped the fractions that contained RsbW. Reconstitution experiments were performed in which partially purified oB and RsbW were added to core RNA polymerase and tested for their ability to influence the transcription of a oB-dependent promoter (ctc) in vitro. RsbW efficiently blocked oB-dependent transcription but only if it was incubated with aB prior to the addition of the core enzyme.