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

Abstract

ABSTRACT The C-terminal domain of the α subunit (αCTD) of Escherichia coli RNA polymerase is often involved in transcriptional regulation. The αCTD 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 αCTD 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 αCTD or His-α proteins were substituted for RNA polymerase. In gel mobility shift assays, the formation of a supershifted ternary complex demonstrated that Mor and His-α bind synergistically to P m DNA. Gel shift assays with short DNA fragments demonstrated that only the Mor binding site and a single upstream αCTD binding site were required for ternary complex formation. These results suggest that the αCTD 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.