Structure of a Bacillus subtilis bacteriophage SPO1 gene encoding a RNA polymerase af factor

Gene 28 of Bacillus subtilis bacteriophage SPOl codes for a regulatory protein, a a factor known as oroz's that binds to the bacterial core RNA polymerase to direct the recognition of phage middle gene promoters. middle promoters exhibit distinctive and conserved nucleotide sequences in two regions centered about 10 and 35 base pairs upstream from the start point of mRNA synthesis. Here we report the cloning of gene 28 and its complete nucleotide sequence. We infer that oa9028 is a 25,707dalton proteinof 220 amino acids. Neither the nucleotide sequence of gene 28 nor the inferred amino acid sequence of co 028 exhibits extensive homology to the gene or protein sequence ofEscherichia coli a factor. The a subunit of bacterial RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) plays a critical role in the initiation of transcription (1). The binding of 0to the core enzyme confers on polymerase the ability to recognize promoter sites on DNA which, in Escherichia coli, exhibit characteristic conserved sequences in two regions centered about 10 base pairs (bp) (the -10 region) and 35 bp (the -35 region) upstream from the start point of transcription. The consensus sequences on the nontranscribed strand for the -10 and -35 regions are T-A-T-A-A-T and T-T-G-A-C-A, respectively (2, 3). In E. coli, a single 70,263-dalton a factor is thought to initiate all transcription (4). In Bacillus subtilis and its bacteriophages, in contrast, multiple species of 0factor confer upon core RNA polymerase the ability to bind to different classes of promoters (5). RNA polymerase containing a55, the principal a0factor used by B. subtilis during vegetative growth, recognizes promoters that are similar in sequence to E. coli promoters (6, 7). During the lytic growth of certain bacteriophages and during the process of endospore formation, 0-51 is replaced on the core RNA polymerase by novel phage and bacterial species of a. The prototype for studies of novel 0factors is the product of bacteriophage SPO1 gene 28, a regulatory protein known as ugP28, which directs the recognition of phage middle gene promoters at intermediate times during the SPO1 lytic cycle (8-10). Like E. coli promoters or promoters recognized by the principal form of B. subtilis RNA polymerase, middle gene promoters strongly conform to each other in two sequences centered at positions -35 and -10. The conserved -35 and -10 regions of middle gene promoters (A-G-G-A-G-A and T-T-T-NT-T-T, respectively; T represents hydroxymethyluracil in SPO1 DNA) differ significantly, however, from the corresponding canonical hexamers of E. coli promoters or B. subtilis ar55-controlled promoters (11, 12). Interestingly, this pattern of distinctive and conserved -10 and -35 regions is emerging as a general feature of promoters controlled by the various bacterial and phage-coded a-s in B. subtilis (13-15). This argues that RNA polymerases with different species of a factor see promoters in fundamentally the same manner but that different 0-factors interact with different bases in both the -35 and -10 regions (5, 12). How then do the structures of these novel os compare with each other or with the structure of E. coli a? The primary structure of E. coli a-has been derived from the nucleotide sequence of its cloned gene rpoD (4). Until now, the primary structure of any a factor acting in B. subtilis has been unknown. Here we report the cloning of SPO1 gene 28, its complete nucleotide sequence, and the derived primary structure of a-g28. MATERIALS AND METHODS Construction of Recombinant Plasmids. SPOI phages were grown and purified as described (8); gene 28 mutants susF21 and sus33 were provided by S. Okubo (16). Phage and plasmid DNAs were purified and cleaved with restriction enzymes, and their fragments were isolated as described (17, 18) except that DNA fragments were eluted from agarose gels by the method of Vogelstein and Gillespie (19). Sau3A-cleaved EcoRI* fragment 3 (2 jig) isolated from either wild-type SPO1 DNA (to construct pMC103) or gene 28 mutant susF21 DNA (to construct pMC2) was cloned into BamHIcleaved pBR322 (0.2 jig). Conditions for in vitro ligation, transformation, and preparation of colony hybridization filters were as described (20) except that T4 DNA ligase was purchased from Bethesda Research Laboratories and E. coli strain HB101 was the transformation recipient. DNA for use as a hybridization probe (see Results) was radioactively labeled by nick-translation (21). Hybridizations were performed as described (18) except that a 200-fold excess of unlabeled pBR322 DNA was added to the hybridization mixture for pM.C103 screening. In Vitro Transcription-Translation. Coupled in vitro transcription-translation was performed with an S150 extract, ribosomes, and initiation factors purified from E. coli MRE 600 cells according to the method of-Schweiger and Herrlich (22). DNA (1-2 Ag) was used in a 50-,ud reaction mixture containing 15 ,uCi (1 Ci = 3.7 X 1010 Bq) of [3S]methionine (from New England Nuclear). Samples were precipitated with trichloroacetic acid, resuspended in sample buffer, and run on 12% poly*acrylamide gels by the method of Laemmli (23). DNA Sequence Determination. Restriction fragments were radioactively labeled at their 5' ends by the phosphate exchange kinase reaction with T4 polynucleotide kinase (from Boehringer Mannheim) (24) or at their 3' ends by extension with the Klenow fragment of E. coli DNA polymerase I (from Bethesda Research Laboratories) (25). Nucleotide sequence was determined as described by Maxam and Gilbert (26). Abbreviations: bp, base pair(s); kb, kilobase(s). 1236 The publication costs of this article were defrayed in part by page charge payment. This article must therefore. be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 80 (1983) 1237 Purification and NH2-Terminal Amino Acid Sequence of gp28. A plasmid capable of overproducing gp28 in E. coli was constructed by ligating the Sau3A insert from pMC103 into the BamHL site of pA8, a pBR322 derivative containing a 1.1-kilobase (kb) fragment of bacteriophage A DNA bearing the strong promoter PL and the N gene (27). Induction of transcription from PL, by inactivation of the temperature-sensitive A repressor carried by the host strain, resulted in the production of gp28 as a significant-fraction of total cell protein. gp28 was purified to apparent homogeneity from extracts of induced cells by DEAESephacel and Sephacryl S-200 chromatography. The sequence of the first five NH2-terminal amino acids of gp28 was determined on a Beckman sequencer with approximately 50 Ag of gp28. The overproduction, purification, and NH2-terminal amino acid sequence of gp28 will be described in detail elsewhere.