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The Bacteriophage T4 Transcription Activator MotA Interacts with the Far-C-Terminal Region of the ⁷⁰ Subunit of Escherichia coli RNA Polymerase

Suchira Pande,^1, Anna Makela,¹ Simon L. Dove,^2, Bryce E. Nickels,² Ann Hochschild,² and Deborah M. Hinton^1*

Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0830,¹ Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115²

Received 23 July 2001/ Accepted 24 April 2002

Transcription from bacteriophage T4 middle promoters uses Escherichia coli RNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA. AsiA binds tightly within the C-terminal portion of the ⁷⁰ subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at -30, and also interacts with ⁷⁰. We show here that the N-terminal half of MotA (MotA^NTD), which is thought to include the activation domain, interacts with the C-terminal region of ⁷⁰ in an E. coli two-hybrid assay. Replacement of the C-terminal 17 residues of ⁷⁰ with comparable ³⁸ residues abolishes the interaction with MotA^NTD in this assay, as does the introduction of the amino acid substitution R608C. Furthermore, in vitro transcription experiments indicate that a polymerase reconstituted with a ⁷⁰ that lacks C-terminal amino acids 604 to 613 or 608 to 613 is defective for MotA-dependent activation. We also show that a proteolyzed fragment of MotA that contains the C-terminal half (MotA^CTD) binds DNA with a K_D(app) that is similar to that of full-length MotA. Our results support a model for MotA-dependent activation in which protein-protein contact between DNA-bound MotA and the far-C-terminal region of ⁷⁰ helps to substitute functionally for an interaction between ⁷⁰ and a promoter -35 element.

Present address: SHU-CHI LLC, Fairfax, VA 22031.

Present address: Division of Infectious Diseases, The Children's Hospital, Harvard Medical School, Boston, MA 02115.

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