In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing sigmaA, the major sigma factor in growing cells.
Myxococcus xanthus is a Gram-negative bacterium that undergoes multicellular development upon starvation. We have developed a simple and rapid procedure for partial purification of RNA polymerase from growing M. xanthus cells, using heparin-agarose and DNA-cellulose chromatographies. In addition to core subunits, the enzyme contains one fairly abundant polypeptide of approximately 105 kDa. We have shown by Western blot analysis and protein sequencing that the 105-kDa polypeptide is sigmaA, the product of the M. xanthus sigA gene. Partially purified sigmaA RNA polymerase, or holoenzyme reconstituted from sigmaA and core RNA polymerase, transcribed in vitro the vegA and aphII genes that are known to be expressed in growing M. xanthus cells. Reconstituted sigmaA RNA polymerase produced vegA mRNA in vitro with the same 5' end as vegA mRNA produced in vivo, demonstrating that initiation of transcription was accurate in vitro. These results provide biochemical evidence that sigmaA is the major vegetative sigma factor of M. xanthus. To our knowledge, this is the first report of in vitro transcription of M. xanthus chromosomal genes, providing a foundation for further biochemical analysis of transcriptional regulatory mechanisms in a microbe that relies extensively on cell-cell interactions.
Biran
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D
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Kroos
L
L
D000907:Antibodies, Bacterial; D001412:Bacillus subtilis / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D005798:Genes, Bacterial; D006657:Histones; D016941:Myxococcus xanthus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D006657:Histones; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012808:Sigma Factor; C054242:VegA protein, Myxococcus xanthus; C063505:RNA polymerase sigma A; D012321:DNA-Directed RNA Polymerases