An RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) has been purified from phage-SPOl-infected Bacillus subtilis that copies RNA almost exclusively from the heavy strand of native SPOl DNA, the DNA strand from which "middle" and "late" classes of RNA are copied in vivo. Hybridization-competition established that this RNA polymerase, termed enzyme A, preferentially synthesizes middle RNA in vitro. Enzyme A contains $', , a, and two newly identified host polypeptides, a (21,500 daltons) and co (11,000 daltons). All of these polypeptides are associated with highly purified RNA polymerase from uninfected bacteria.In addition, enzyme A contains phage-induced subunits of 26,000, 24,000, and 13,500 daltons. Enzyme A lacks a-polypeptide, and strand-selective transcription by this enzyme is resistant to anti-a antibody. A reconstitution experiment strongly suggests that the host 5 protein is required in addition to a phage-induced subunit(s) (or an unidentified phage-induced modification) for strand-selective transcription of SPOl middle genes in vitro.The Bacillus subtilis DNA phage SP01 directs the synthesis of six different classes of phage-specified transcripts in a temporally defined sequence (1). Two classes of early RNA (e and em) appear immediately after infection and are copied from both the heavy (H) and light (L) strands of SP01 DNA. This transcription is not inhibited by chloramphenicol and presumably does not require phage-specified protein synthesis. About 4-5 min after infection two classes of middle transcripts (m and mil) are first synthesized largely from strand H (1). This gene transcription requires the protein product of regulatory gene 28 (2, 3). Finally, late RNA transcripts (m21 and 1) do not appear until 8-14 min into the lytic cycle and are transcribed almost exclusively from strand H. Mutations in regulatory genes 33 and 34 block the synthesis of late RNA (1-3).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. In this paper, we report on the purification of an RNA polymerase from infected cells containing phage-induced subunits and a newly identified host polypeptide of 21,500 daltons. This enzyme (enzyme A) selectively transcribes middle genes from the H strand of SP01 DNA, whereas, RNA polymerase from uninfected cells transcribes early genes from both strands of SP01 DNA. As will be discussed, enzyme A is apparently different from other RNA polymerases isolated from phage-infected B. subtilis that have been reported to synthesize middle RNA in titro (5-7). METHODSPreparation of SPOl DNA. Wild-type SP01 (obtained from D. Shub) was grown and partially purified as described (8) except that phage were initially precipitated with polyethylene glycol. Phage were further purified on a CsCl step grad...

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BOTANY): PARTANEN AlND NELSON 1165 trations used in these studies, however, the optical rotation of the coenzymne in phosphate buffer, employed as a polarimeter blank, is barely detectable.