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Binding of the C-Terminal Domain of the Subunit of RNA Polymerase to the Phage Mu Middle Promoter

Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee

Received 31 March 2004/ Accepted 27 August 2004

The C-terminal domain of the subunit (CTD) of Escherichia coli RNA polymerase is often involved in transcriptional regulation. The CTD typically stimulates transcription via interactions with promoter UP element DNA and transcriptional activators. DNase I footprinting and gel mobility shift assays were used to look for potential interaction of the CTD with the phage Mu middle promoter P_m and its activator protein Mor. Binding of RNA polymerase to P_m in the presence of Mor resulted in production of a DNase I footprint downstream of Mor due to open complex formation and generation of a second footprint just upstream of the Mor binding site. Generation of the upstream footprint did not require open complex formation and also occurred in reactions in which the CTD or His- proteins were substituted for RNA polymerase. In gel mobility shift assays, the formation of a supershifted ternary complex demonstrated that Mor and His- bind synergistically to P_m DNA. Gel shift assays with short DNA fragments demonstrated that only the Mor binding site and a single upstream CTD binding site were required for ternary complex formation. These results suggest that the CTD plays a role in P_m transcription by binding to P_m DNA just upstream from Mor and making protein-protein interactions with Mor that stabilize the binding of both proteins.