RNA polymerase of Myxococcus xanthus: purification and selective transcription in vitro with bacteriophage templates

Abstract

DNA-dependent RNA polymerase from vegetative cells of the gram-negative, fruiting bacterium Myxococcus xanthus was purified more than 300-fold by a modified Burgess procedure (Lowe et al., Biochemistry 18:1344-1352), using Polymin P precipitation, 40 to 65% saturated ammonium sulfate fractional precipitation, double-stranded DNA cellulose chromatography, A5m gel filtration chromatography, and single-stranded DNA agarose chromatography. The last step separated the RNA polymerase into a core fraction and an enriched holoenzyme fraction. The core enzyme showed a subunit structure similar to that of the Escherichia coli polymerase, as follows: 1' and , (145,000 and 140,000 daltons, respectively) and a (38,000 daltons). A comparison of the core enzyme and the holoenzyme implicated two polypeptides as possible a subunits. These polypeptides were closely related, as indicated by peptide analysis. M. xanthus RNA polymerase was capable of transcribing DNAs from E. coli phages T7, T4, and X, Bacillus subtilis phage 4)29, and M. xanthus phages Mxl, Mx4, and Mx8. Transcription of T7 and 4)29 DNAs was stimulated by KCl, whereas transcription of Mxl, Mx4, and Mx8 DNAs was inhibited by KCI. Magnesium ion dependence, rifampin and heparin sensitivities, and spermidine stimulation of M. xanthus RNA polymerase activity were similar to those found with E. coli RNA polymerase. The pH optimum of M. xanthus RNA polymerase activity was more basic than that of E. coli polymerase. M. xanthus RNA polymerase was capable of selective transcription in vitro when DNAs from phages T7 Alit, 4)29, and Mxl were used. The molecular weights of the resulting phage RNA transcripts made by M. xanthus RNA polymerase (as determined by agarose-acrylamide slab gel electrophoresis) were the same as the molecular weights of the transcripts synthesized by E. coli RNA polymerase. No discrete transcripts were detected as the in vitro RNA products of M. xanthus phage Mx4 and Mx8 DNA transcription. Southern blot hybridization analysis was used to confirm the identity of the T7 ADlll A1 transcript synthesized by M. xanthus RNA polymerase. Three transcripts (trant Present address:

In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing σA, the major sigma factor in growing cells

“…1). The putative SigA appeared to be fairly stable when cells were lysed in the presence of protease inhibitors as described previously (Rudd and Zusman, 1982); however, a slightly smaller polypeptide was the predominant species after Polymin P precipitation and ammonium sulphate fractionation (data not shown), suggesting that proteolysis was occurring during these early purification steps. When we used heparin-agarose chromatography instead, the 105-kDa polypeptide was stabilized for further purification (Figs 2 and 3).…”

“…M. xanthus cells secrete proteases and use the resulting amino acids as carbon and energy sources, as well as substrates for protein synthesis (Dworkin, 1962;Bretscher and Kaiser, 1978). Rudd and Zusman (1982) reported previously that protease activity presented a substantial problem in recovering active RNA polymerase from growing M. xanthus, but they developed a modified Burgess procedure that allowed the recovery of active enzyme. This procedure employs Polymin P precipitation and fractional ammonium sulphate precipitation to isolate RNA polymerase from the bulk of cellular nucleic acid and protein.…”

“…To purify RNA polymerase partially from vegetative M. xanthus cells, we initially followed a modified Burgess procedure (Lowe et al, 1979) developed by Rudd and Zusman (1982). Proteolysis was a significant problem, as noted previously (Rudd and Zusman, 1982). In particular, degradation of the putative SigA to a slightly smaller form occurred during the early steps of purification (Polymin P precipitation and ammonium sulphate fractionation), as determined by Western blot analysis of fractions with anti-43 antibodies (data not shown).…”

“…To purify RNA polymerase partially from vegetative M. xanthus cells, we initially followed a modified Burgess procedure (Lowe et al, 1979) developed by Rudd and Zusman (1982). Proteolysis was a significant problem, as noted previously (Rudd and Zusman, 1982).…”

“…Despite considerable knowledge about the M. xanthus cell-cell signals and emerging insights into the signal transduction mechanisms, very little is known about the transcriptional machinery that controls gene expression in response to signals. Rudd and Zusman (1982) were the first to describe the isolation of RNA polymerase from growing M. xanthus cells. In addition to core enzyme subunits (␣, ␤ and ␤Ј) typical of prokaryotes, two possible subunits were present in holoenzyme fractions.…”