Binding of the C-terminal domain of the alpha subunit of RNA polymerase to the phage mu middle promoter.
The C-terminal domain of the alpha subunit (alpha CTD) of Escherichia coli RNA polymerase is often involved in transcriptional regulation. The alpha 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 alpha 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 alpha CTD or His-alpha proteins were substituted for RNA polymerase. In gel mobility shift assays, the formation of a supershifted ternary complex demonstrated that Mor and His-alpha bind synergistically to P(m) DNA. Gel shift assays with short DNA fragments demonstrated that only the Mor binding site and a single upstream alpha CTD binding site were required for ternary complex formation. These results suggest that the alpha 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.
Ma
Ji
J
Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Ave., Memphis, TN 38163, USA.
Howe
Martha M
MM
D010583:Bacteriophage mu / Q000235:genetics*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
C101385:Mor protein, Enterobacteria phage Mu; D015534:Trans-Activators; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit; D003850:Deoxyribonuclease I