RNA polymerase from phage SP01-infected and uninfected Bacillus subtilis.
A purification procedure for RNA polymerase from uninfected and phage SP01-infected Bacillus subtilis is presented. The RNA polymerase purified from B. subtilis 10 min after infection with wild type phage SP01 is resolved into two major fractions (B, C) and one minor fraction (A) by calf thymus DNA-cellulose chromatography. Fraction C is indistinguishable from RNA polymerase from uninfected cells with respect to transcription specificity (both before and after phosphocellulose chromatography). Fraction B yields, on subsequent phosphocellulose chromatography, an enzyme (B-P) whose properties distinguish it from the host RNA polymerase. Enzyme B-P preferentially transcribes SP01 DNA and selectively forms rapidly initiating complexes with SP01 DNA but not with heterologous DNA. The SP01 RNA synthesized by Enzyme B-P includes, as previously reported, a large proportion of asymmetrical middle viral RNA. Host RNA polymerase holoenzyme synthesizes asymmetrical early viral RNA, while host core polymerase synthesizes symmetrical RNA that is complementary to early, middle, and late in vivo viral RNA and contains a preponderance of antimessenger. The subunit composition of Enzyme B-P is identical to host core polymerase with respect to the beta,beta', and alpha subunits and two additional components of mr equals 9,500 and 11,000 that we observe in all preparations of RNA polymerase. In addition, Enzyme B-P has two subunits of mr equals 13,000 and 28,000, which are synthesized after phage infection. On heterologous template, Enzyme B-P and host core polymerase have comparable activities. On these templates, addition of host initiation factor, sigma, restores full activity to Enzyme B-P as well as to host core polymerase. Sigma also modifies the activity of Enzyme B-P on SP01 DNA, restoring some asymmetrical early RNA transcription while retaining some asymmetrical middle RNA transcription.
D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology*; D001435:Bacteriophages / Q000201:enzymology*; D002417:Cattle; D002455:Cell Division; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004247:DNA; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D013950:Thymus Gland; D013997:Time Factors; D014158:Transcription, Genetic
D004247:DNA; D012321:DNA-Directed RNA Polymerases; D007930:Leucine