CONFORMATIONAL PROPERTIES OF BACILLUS-SUBTILIS RNA-POLYMERASE SIGMA(A)-FACTOR DURING TRANSCRIPTION INITIATION

By the use of a partial proteolysis method and Western-blot analysis, the conformational properties of Bacillus subtilis sigma(A) factor in the transcription initiation stage were studied. From a comparison of the trypsin-digestion patterns of free sigma(A) and of sigma(A) associated with core enzyme, it was found that the production of 45 kDa sigma(A) tryptic-derived fragment was enhanced when e was associated with the core enzyme. More importantly, a 40 kDa sigma(A) tryptic-derived fragment was found exclusively in this associated state. Based on the change of the digestion kinetics when producing the 45 kDa tryptic fragment and the generation of this new 40 kDa tryptic fragment from sigma(A), it was apparent that a conformation change of sigma(A) occurred during the association of sigma(A) with the core enzyme. Also, similar patterns were found for the e present in the holoenzyme-promoter DNA complex. These findings suggest that no further distinctive conformational change of sigma(A) occurs at the step of RNA polymerase holoenzyme and promoter DNA complex formation. Trypsin-digestion patterns of sigma(A) in different RNA polymerase holoenzyme and promoter DNA complexes were also studied. The presence of similar trypsin digestion-patterns of sigma(A) in those complexes strongly supports the idea that a similar sigma(A) conformation is used in the recognition of different sigma(A)-type promoters and the formation of different open complexes.