Subtilis Sigma Research Articles

Peptide mapping of Bacillus subtilis RNA polymerase alpha factors and core-associated polypeptides.

An analysis of the peptide maps of the sigma factors and core-associated subunits of Bacillus subtilis RNA polymerase has revealed that all the sigma factors ad core-associated polypeptides are derived from separate genes and are not proteolytically modified products of the major 55,000-dalton sigma factor. A comparison of the peptide pattern of the major B. subtilis and Escherichia coli sigma factors revealed limited homology between them. Furthermore, antibody prepared against the 55,000-dalton B. subtilis sigma factor cross-reacted against the E. coli sigma factor, but not against any of the other B. subtilis sigma factors and core-associated polypeptides. These results unambiguously demonstrate the independently derived nature of the B. subtilis RNA polymerase core-associated subunits and the partial relationship between the major sigma factors of B. subtilis and E. coli.

Promoter for a developmentally regulated gene in Bacillus subtilis

We have determined the nucleotide sequence of the promoter for a B. subtilis gene (the 0.4 kb gene) whose transcription is under developmental control. Transcription of the 0.4 kb gene is turned on at the onset of sporulation; this RNA synthesis depends on the products of the B. subtilis regulatory genes (the spoO loci) that control the initiation of development. Recognition of the 0.4 kb gene promoter in vitro is dictated by novel species of B. subtilis RNA polymerase sigma factor known as σ 37 and σ 29 but not by the principal B. subtilis sigma factor σ 55. Using S1 nuclease mapping, runoff transcription and dinucleotide priming, we have identified dual startpoints (separated by about 10 bp) for σ 37-directed transcription of the 0.4 kb gene. These startpoints correspond closely to the 5′ termini of 0.4 kb RNA synthesized in vivo during the course of sporulation. Two forms of σ 37-containing RNA polymerase were distinguished that preferentially utilize either the upstream or the downstream startpoint in vitro. We investigated the requirements for σ 37-directed transcription by constructing in vitro deletion mutations that extend from the upstream direction into the 0.4 kb promoter region. One such deletion, which terminates 40 and 51 bp upstream from the transcription startpoints (thereby removing a highly AT-rich 26 bp sequence), completely eliminates transcription from the downstream startpoint but only partially inhibits transcription from the upstream startpoint. A second deletion that terminates at the upstream startpoint completely prevents transcription from both initiation sites. The implications of 0.4 kb gene promoter structure for developmentally regulated transcription in B. subtilis are discussed.

Free sigma subunit of Bacillus subtilis RNA polymerase binds to DNA

The affinity of Bacillus subtilis RNA polymerase sigma and delta subunits to DNA was examined by a non-denaturing polyacrylamide slab gel electrophoresis method which made it possible to resolve DNA-bound and free subunits. The results revealed that sigma subunit, but not delta subunit had a relatively high affinity for double stranded DNA. The sigma subunit was bound maximally to super-coiled pGR1-3 plasmid DNA at a mass ratio of sigma/DNA of 0.7. With B. subtilis double stranded linear DNA one sigma subunit was bound per approximately 1,000 base pairs. The sigma-DNA complex was sufficiently stable for isolation by a molecular gel filtration column. The sigma subunit had much higher affinity for super-coiled than for linear pGR1-3 DNA or for linear double stranded or denatured DNA from B. subtilis, E. coli, and calf thymus. These results indicate that the free B. subtilis sigma subunit, in contrast to the E. coli sigma subunit, can bind by itself to DNA.

Nucleotide sequence of a Bacillus subtilis promoter recognized by Bacillus subtilis RNA polymerase containing sigma 37.

We report the nucleotide sequence of the promoter for a Bacillus subtilis gene (spoVC) whose transcription is controlled by a 37,000 dalton species of B. subtilis sigma factor known as sigma 37 but not by the principal- sigma factor of 55,000 daltons (sigma 55). Using S1 nuclease mapping we show that the startpoint for sigma 37-directed transcription of the spoVC gene in vitro corresponded closely to the 5' terminus of in vivo synthesized spoVC RNA. The binding site for sigma 37-containing RNA polymerase extended from 43 bp to 51 bp (positions -43 to -51) upstream from the transcription startpoint to 22 bp (position +22) downstream from the startpoint. The nucleotide sequence of the spoVC promoter differed significantly from promoters whose recognition is controlled by sigma 55 but was similar to other sigma 37- controlled promoters in regions known to be important in promoter recognition. Our results are consistent with the hypothesis (Lee and Pero, J. Mol. Biol., in press) that sigma factors work by contacting specific bases in both the -35 and -10 regions of promoters.

The interaction of Escherichia coli core RNA polymerase with specificity-determining subunits derived from unmodified and SP82-modified Bacillus subtilis RNA polymerase.

the ability of the core isolated from Escherichia coli RNA polymerase to interact with specificity-determining subunits isolated from Bacillus subtilis RNA polymerase has been determined by measuring the transcription of "early" and "middle" genes of phage SP82. Two specificity-determining subunits were tested: the sigma subunit and a 28,000 dalton (28 K) peptide isolated from a modified polymerase produced at approximately 8 min after infection of B. subtilis with SP82. Earlier experiments (Spiegelman, G. B. and Whiteley, H. R. (1978) Biochem. Biophys. Res. Commun. 81, 1058-1065) demonstrated that sigma and the 28K peptide are required for the recognition of early and middle gene promoters, respectively, by the B. subtilis core assembly. The present investigation showed that E. coli core interacted more efficiently with the B. subtilis sigma than with the 28K peptide, as judged by the rate of RNA synthesis. Early RNA was produced by the E. coli and B. subtilis holoenzymes and by E. coli core supplenented with B. subtilis sigma and only minor differences were found in comparisons of transcripts by hybridization and by electrophoretic analysis. Measurements of template specificity, the formation of stable enzyme . DNA complexes, and the hybridization of transcripts to fragments of SP82 DNA produced by digestion with restriction endonuclease Hha indicated that E. coli core supplemented with the 28K-supplemented E. coli core with those synthesized by the modified polymerase extracted from B. subtilis 8 min after infection with SP82 suggest that both preparations recognized the same initiation and termination sequences.

Altered promoter selection by a novel form of Bacillus subtilis RNA polymerase.

Bacillus subtilis RNA polymerase holoenzyme prepared by several standard methods utilizes bacteriophage T7 DeltaD111 DNA as an efficient template. The major RNA products are specific transcripts from T7 promoters A(1) and C; these promoters are also efficiently utilized by RNA polymerases purified from a wide range of other bacterial species [Wiggs, J., Bush, J. & Chamberlin, M. (1979) Cell 16, 97-109]. In contrast, B. subtilis RNA polymerase preparations purified by a modification of the method of Burgess and Jendrisak (designated fraction 5) utilize T7 DeltaD111 promoters A(1) and C and an additional promoter site, J, which has been located at 90.6% on the standard T7 physical map. This promoter is not used by B. subtilis core RNA polymerase or by RNA polymerase from any other bacterial species we have tested. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of fraction 5 RNA polymerase shows that it contains B. subtilis components sigma and delta and a polypeptide of M(r) 92,000 in addition to the B. subtilis beta, beta', and alpha subunits. Chromatography of fraction 5 on single-stranded DNA-cellulose gives an enzyme fraction, Bs I, that is indistinguishable from B. subtilis RNA polymerase holoenzyme both in its peptide composition (betabeta'alpha(2)sigma) and in the selective transcription of only T7 RNAs A(1) and C. Chromatography of fraction 5 on phosphocellulose yields an enzyme fraction, Bs II, devoid of sigma subunit but containing the M(r) 92,000 peptide and traces of delta. This fraction synthesizes predominantly T7 J RNA in vitro together with traces of T7 A(1) and C RNAs. Hence, B. subtilis RNA polymerase fraction Bs II appears to contain a form of RNA polymerase that can transcribe selectively without detectable amounts of B. subtilis sigma subunit and that utilizes a promoter site not used by other known bacterial RNA polymerases. The structural basis for this specificity is not yet known.

Comparative Size and Properties of the Sigma Subunits of Ribonucleic Acid Polymerase from Bacillus subtilis and Escherichia coli

Bacillus subtilis RNA polymerase transcribes phage Oe DNA more actively than phage T4 DNA, while Escherichia coli RNA polymerase is equally active with both templates. This paper investigates the role of sigma in determining this difference in template specificity by in vitro complementation of B. subtilis core polymerase and E. coli core polymerase with both the homologous and the heterologous sigma subunits. Each core polymerase retained its characteristic template specificity in the presence of either sigma subunit. To determine whether the smaller size of B. subtilis sigma (55,000 daltons) compared to that of E. coli sigma (86 to 95,000 daltons) is due to in vitro proteolysis, RNA polymerase was purified from a mixture of E. coli and B. subtilis separately labeled with different radioisotopes. Sodium dodecyl sulfate gel electrophoresis of the purified polymerases demonstrated that the two sigma subunits retained their characteristic molecular weights when purified from the mixture.