Chapter 10 : Transcription Regulation by the Bacillus subtilis Response Regulator Spo0A

Amino acid sequence of Spo0A. The amino acid sequence of Spo0A is taken from Ferrari et al. (1985) .Two regions of the amino acid sequence of OmpR are shown where transcription activation mutants (underlined) have been identified ( Russo et al., 1993 ). At the C terminus, the amino acid sequence from ϕ29 gp4 and various mutations of the sequence that affect the activity of the protein are shown. The sequence is from Mizukami et al. (1986) and the mutants are from Mencia et al. (1993) and Rojo et al. (1990) . Positions of mutations in Spo0A are shown: sof and sob mutants are from Spiegelman et al. (1990) , sad mutants that are deletions of the indicated amino acids are from Ireton et al. (1993) , the coi mutants are from Olmedo et al. (1990) and Δ209, Δ267 mutants are from Green et al. (1991) . The DNA binding motif in Spo0A proposed by Youngman (personal communication) is underlined. Regions marked as helical (α) and extended (β) were calculated using the GGBSM program of PC gene. These regions are not meant to be precise but to allow subdivision of the sequence for discussion. Regions were calculated for the N-terminal and C-terminal domains separately, and so they begin again after the diamond symbol, which indicates the division between the N-terminal and C-terminal domains (as defined in the text). In the C-terminal domain, the positions of the spo0A9 and spo0A153 mutations are indicated (at position 257), and the positions of suppressors of spo0A9V are indicated at H162F (suv4) and L174F (suv3) ( Perego et al., 1991 ).

Formation of initiation complexes at spoIIGp: stimulation by Spo0A, Spo0A-P, and Spo0ABD. Transcription initiation reactions were composed and analyzed as described in Bird et al. (1992) . Reactions contained 100 nM Spo0A-P (closed circles), Spo0ABD (triangles), or Spo0A (open circles), GTP, ATP, and a DNA fragment containing spoIIGp At time zero, RNA polymerase was added to the reaction, and after various incubation times, samples were withdrawn and challenged with a mixture containing CTP, UTP, and heparin. The percentage of template containing an initiated complex is plotted as a function of the time of the initiation reaction assay. The reaction containing Spo0A-P initiated very rapidly, whereas those containing the binding domain showed a pronounced lag, although the final level was only slightly lower than that seen with Spo0A-P.

0A boxes in regulated promoters. (A) Sequences that have been identified as being protected by Spo0A from digestion by DNase I are indicated in capital letters. In some sequences, the protected regions are not flush on both strands of DNA and unprotected DNA is indicated by lowercase letters. In examples in which both strands are capitals, comparison of the digestion pattern on the two strands has not been performed. The consensus sequence for the 0A box is underlined. The numbering refers to the +1 site for each promoter. (B) Position of the OA boxes relative to the start site of transcription for the promoter is indicated. The symbols indicate forward (>>>>) or reverse (<<<<) orientations. Indicated at the side is the gene or operon where the 0A boxes are located and the σ-factor used by the promoter (σA or σH). Promoters that are activated by Spo0A have an asterisk (*); the other promoters are repressed. The data are taken from spoIIG ( Satola et al., 1991 ; Satola et al. , 1992 ; Baldus et al., 1994 ),spoIIE ( York et al., 1992 ), spoIIA ( Trach et al., 1991 ; Baldus et al., 1994 ),spo0A ( Strauch et al., 1992 ), spo0F ( Strauch et al., 1993 ), and abrB ( Strauch et al., 1990 ).

Repression of transcription of abrBp 1 and abrBp 2 promoters by (A) Spo0A-P and (B) Spo0ABD. An 800-bp HindIII-EcoRI fragment from PJM5134 ( Perego et al., 1988 ) was excised and filled with the Klenow fragment to create blunt ends. This fragment was used as the template (at 5 nM) in transcription assays composed as described in Bird et al. (1993) . The transcription products were separated by electrophoresis through polyacrylamide gels containing 7 M urea and localized and quantitated as described in Bird et al. (1993) . The results are presented as the fraction of the extent of transcription in the absence of either Spo0A-P or Spo0ABD. Spo0A-P or Spo0ABD were preincubated with the template at 37°C. RNA polymerase was added, and after 3 min, nucleotides allowing transcript elongation were added. After a 3-min initiation-elongation reaction, heparin was added. This is a multiple round initiation assay. Open symbols are transcripts from abrBp 1 ; closed symbols are from abrBp 2 .

Stimulation of transcription from spoIIAp by Spo0A-P and σH. The template used contained spoIIAp cloned adjacent to ribosomal RNA terminators, and the transcription reaction conditions and analysis of product have been described ( Bird et al., 1992 ). Core RNA polymerase was incubated with the DNA template, increasing amounts of recombinant σH, either without Spo0A (triangles) or with Spo0A-P (circles). As seen in the presence of Spo0A-P, the stimulation of transcription by low levels of the σ-factor was enhanced. At higher inputs of σH, the increase in transcription was the same in the presence and absence of Spo0A-P. Thus, the presence of Spo0A-P increased the stimulation of transcription by the σ-factor.

DNase I footprint assay of formation of complexes at spoIIGp. A HindIII to PvuII DNA fragment containing spoIIGp ( see Bird et al., 1993 ) was labeled at the HindIII end with [γ-32P]ATP and polynucleotide kinase. The fragment was incubated in transcription reaction buffer either alone or in the presence of either Spo0A or Spo0A-P. All reactions contain 0.4 mM ATP, which allows formation of a dinucleotide at spoIIGp. An aliquot of each reaction was removed and treated with 4 µg/ml DNase I for 10 s. The reaction was stopped with 10 mM EDTA, 0.1% sodium dodecyl sulfate. Labeled DNA was recovered by ethanol precipitation, redissolved in formamide gel loading buffer, and electrophoresed on a 6% polyacrylamide gel containing 7 M urea. To examine the rate of complex formation, RNA polymerase (100 nM, final concentration) was added to the template, and after different incubation times, samples were removed and treated with DNase for 10 s as described above. The incubation times (after adding the polymerase) are lane 1,5 s; lane 2, 30 s; lane 3, 1 min; lane 4, 2 min; lane 5, 5 min, lane 6, 15 min. For each set, the lane marked C shows the pattern obtained when no proteins were added, and the lane marked 0 is the pattern obtained before adding RNA polymerase. The nucleotide positions, relative to the transcription initiation site, are given at the left. Without Spo0A or Spo0A-P, a complex between the polymerase and the DNA was formed very rapidly. From our kinetic data, this complex (C) cannot initiate RNA synthesis when challenged with nucleotides and heparin and therefore represents a heparin-sensitive complex. When Spo0A was present, RNA polymerase formed a second type of complex (CII), as indicated by the DNase I-hypersensitive sites (arrowheads at the right). This complex also cannot initiate RNA synthesis rapidly when challenged with heparin and thus represents a second heparin-sensitive complex. In the presence of Spo0A-P, a complex (CIII) formed that lacked one of the sites hypersensitive to DNase I and showed increased protection in the -10 region. This complex cannot complete transcripts when challenged with heparin and nucleotides and so represents a stage before the heparin-resistant complex (HR). The kinetic data suggest that the complex formed in the presence of Spo0A-P (CIII) can initiate RNA synthesis rapidly. We view the CIII complex as equivalent to the activated intermediate in the transition to HR complex (Bird et al., unpublished data).

Reaction mechanism for transcription stimulation at spoIIGp. In the cartoon of initiation, the following symbols are used: double lines represent the DNA template; the large oval represents RNA polymerase; the small circle attached to a small oval represents the two-domain structure of Spo0A. The predicted -10 and —35 consensus sequences at the promoter are indicated on the DNA strands. These sequences are separated by 22 bp instead of the expected 17 to 18 bp. The initial binding of the polymerase and DNA, which is independent of Spo0A, protects only the -35 site of the promoter (CI complex). The binding of Spo0A and RNA polymerase to the DNA creates DNase I-hypersensitive sites in the -27, -28 region of the promoter and increased protection near the —10 region (CII complex). The DNase I-hypersensitive sites are presumed to result from a distortion of the DNA helix, as represented. The binding of two Spo0A proteins is shown because there are two 0A boxes. Phosphorylation of Spo0A (represented by a change in shading pattern) is presumed to cause a change in the shape of the protein. In the presence of Spo0A-P, CII complexes isomerize to CIII complexes, which are characterized by increased protection of the —10 region and loss of the hypersensitive sites. The change is represented by release of the —35 contacts between the polymerase and the promoter, which were presumed to cause the helix distortion. The release of the —35 contacts allows the polymerase to contact the —10 region. The contacts with the -10 region permit the polymerase in the CIII complex to synthesize a short RNA (represented by the thick wavy line) on addition of the initiating nucleotides ATP and GTP. The initiated complex is designated HR because it is resistant to the polymerase inhibitor heparin.