In vitro transcriptional activation of the phage Mu mom promoter by C protein

The phage Mu gene C encodes a 16.5-kDa site-specific DNA-binding protein that functions as a trans-activator of the four phage "late" operons, including mom. We have overexpressed and purified C and used it for DNase I footprinting and transcription analyses in vitro. The footprinting results are summarized as follows. (i) As shown previously (V. Balke, V. Nagaraja, T. Gindlesperger, and S. Hattman, Nucleic Acids Res. 12:2777-2784, 1992) in vivo, Escherichia coli RNA polymerase (RNAP) bound the wild-type (wt) mom promoter at a site slightly upstream from the functionally active site bound on the C-independent tin7 mutant promoter. (ii) In the presence of C, however, RNAP bound the wt promoter at the same site as tin7. (iii) C and RNAP were both bound by the mom promoter at overlapping sites, indicating that they were probably on different faces of the DNA helix. The minicircle system of Choy and Adhya (H. E. Choy and S. Adhya, Proc. Natl. Acad. Sci. USA 90:472-476, 1993) was used to compare transcription in vitro from the wt and tin7 promoters. This analysis showed the following. (i) Few full-length transcripts were observed from the wt promoter in the absence of C, but addition of increasing amounts of C greatly stimulated transcription. (ii) RNA was transcribed from the tin7 promoter in the absence of C, but addition of C had a small stimulatory effect. (iii) Transcription from linearized minicircles or restriction fragment templates was greatly reduced (although still stimulated by C) with both the wt and tin7 promoters. These results show that C alone is capable of activating rightward transcription in vitro by promoting RNAP binding at a functionally active site. Additionally, DNA topology plays an important role in transcriptional activation in vitro.