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Journal of Bacteriology, December 2004, p. 7858-7864, Vol. 186, No. 23
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.23.7858-7864.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Ji Ma and Martha M. Howe*

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 {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 Pm and its activator protein Mor. Binding of RNA polymerase to Pm 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 Pm 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 Pm transcription by binding to Pm DNA just upstream from Mor and making protein-protein interactions with Mor that stabilize the binding of both proteins.

* Corresponding author. Mailing address: Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Ave., Memphis, TN 38163. Phone: (901) 448-8215. Fax: (901) 448-8462. E-mail: mhowe{at}utmem.edu.

Journal of Bacteriology, December 2004, p. 7858-7864, Vol. 186, No. 23
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.23.7858-7864.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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