CHARACTERIZATION OF RNA-POLYMERASE AND 2 SIGMA-FACTOR GENES FROM MYCOBACTERIUM-SMEGMATIS

A search for Mycobacterium smegmatis genes showing similarity to the c onserved family encoding major sigma factors in diverse prokaryotes ha s identified two such determinants. Both genes are expressed in expone ntially growing cells, as judged by Western immunoassays. A series of chromatographic steps was used to purify M. smegmatis RNA polymerase h oloenzyme and it was shown that its ability to initiate in vitro trans cription with a heterologous Bacillus subtilis promoter is dependent o n the presence of these sigma factor(s). Reconstitution of specific in vitro transcription activity was obtained upon mixing of M. smegmatis core RNA polymerase with the major sigma factor of Bacillus subtilis. We also demonstrated in vitro transcription of the M. smegmatis rrnB promoter by the M. smegmatis RNA polymerase. Significantly, highly act ive B. subtilis RNA polymerase holoenzyme was unable to transcribe thi s gene.