New Phage-SPO1-Induced Polypeptides Associated with Bacillus subtilis RNA Polymerase

Abstract

RNA polymerase was precipitated from extracts of Bacillus subtilis infected with phage SPOI by antiserum prepared against core RNA polymerase. As shown by sodium dodecyl sulfate gel electrophoresis, the precipitates contained at least five new polypeptides not present in uninfected bacteria, in addition to the known subunits of RNA polymerase. The molecular weights of these polypeptides are (1) 85,000; (II) 40,000; (II) 28,000; (lV) 25,000; and (V) 23,000. Four of the polypeptides (I, III, IV, and V) co-purified with RNA polymerase through gel filtration and phosphocellulose chromatography. A pulsechase experiment indicated that all five polypeptides are synthesized de novo after infection. The synthesis of polypeptides II, III, and IV commences almost immediately after infection, while polypeptides I and V first appear several minutes later. A sus mutant blocked early in transcription, susF21 [Fujita, et al. (1971) SP01 is a large virulent phage of Bacillus subtilis containing a double-stranded DNA genome of about 108 daltons (1). After infection, SP01 directs the synthesis of six different classes of phage-specified transcripts in a defined temporal sequence (2). This program of phage RNA synthesis requires the rifampicin-sensitive component of the DNA-dependent RNA polymerase of the host bacteria (3) and is controlled by at least three different regulatory genes of the phage (4). However, the molecular mechanism of this regulation is not yet understood.One possible mechanism for the control of phage transcription is by the direct interaction of regulatory proteins of the phage with host RNA polymerase (5). For instance, phage T4 induces four new small polypeptides that bind to Escherichia coli RNA polymerase (6). Two of these proteins have been shown to be the products of regulatory genes 33 (7) and 55 (D. Ratner, personal communication). Also recent reports indicate that RNA polymerase from B. amyloliquefaciens infected with phage 029 contains a new polypeptide of 30,000 daltons (8), and that RNA polymerase from B. subtilis infected with phage SP82 contains at least one new polypeptide of 16,000 daltons (9). However, it is not yet certain whether either of these polypeptides is the product of a regulatory gene.A rapid procedure for isolating RNA polymerase in association with new subunits is by precipitation with antiserum prepared against core RNA polymerase. This method also permits identification of certain subunits of RNA polymerase which dissociate from RNA polymerase during purification. For example, RNA polymerase isolated from sporulating B. subtilis is associated with two new large polypeptides of 70,000 (10) and 95,000 (11, 12) daltons. The 70,000-dalton protein binds to RNA polymerase during precipitation by anti-RNA polymerase antiserum, but it is dissociated from polymerase during phase partitioning (12) or phosphocellulose chromatography (10).To determine whether phage SP01 induces polypeptides that interact directly with B. subtilis RNA polymerase, we have isolated the enzyme f...

Highly asymmetric transcription by RNA polymerase containing phage-SP01-induced polypeptides and a new host protein.

“…Frozen cells (50 g) were disrupted in 300 ml of buffer G (4) by sonication for 20 min at 0-4°. RNA polymerase was first partially purified through the DNase treatment step as described previously (4) except that the dialysis time after RNase (10 mg) treatment was reduced to 4 hr. After DNase (5 mg) treatment (3 hr at 00) insoluble material was removed by centrifugation at 100,000 X g for 1 hr and the supernatant fluid was then directly applied to a column (4 X 80 cm) of Bio-Gel A-1.5 agarose internally supported by 6 mm glass beads.…”

“…RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) isolated from SP01-infected cells is associated with several new phage-induced polypeptides (4). It seemed possible that at least some of these proteins could be regulatory elements that direct the transcription of middle and late genes.…”

Susceptibility of non‐thymine containing DNA to four bacterial restriction endonucleases

“…Phages containing DNA in which hydroxymethyluracil is substituted for thymine are presently being used to study phage development and phage gene expression in sporulating cells in B. subtilis [16][17][18][19][20][21][22]. The availability of specific DNA fragments should be of great use in further analysis of these phage systems; for example, comparing the homology of hydroxymethyluracil containing DNAs and isolation of DNA fragments containing the promotor region~ of phage modified and unmodified RNA polymerases.…”

Conversion of Bacillus subtilis RNA polymerase activity in vitro by a protein induced by phage SP01.

“…Fraction LGG also contains several peptides that are labeled after infection. However, molecular weight coincidence alone is not a strong criterion of identity for comparison with virus-coded proteins that bind to RNA polymerase (9,12,13). Since these peptides in LGG do not bind to RNA polymerase core under our conditions, we cannot comment further on them.…”

“…In contrast, unmodified bacterial RNA polymerase holoenzyme (constitution ,B, ,3', , a, w1, W2) selectively transcribes only the SP01 early genes (8)(9)(10). Spiegelman and Whiteley (11) and Pero and collaborators (12,13) have also found several new peptides bound to RNA polymerase after phage SP82 and SP01 infection and have noted altered transcription specificity. But are these peptides, either singly or together, the determinants of an altered transcription specificity?…”