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Volume 271, Number 50, Issue of December 13, 1996 pp. 32343-32348
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Transcription Activation by the Bacteriophage Mu Mor Protein Requires the C-terminal Regions of Both  and ⁷⁰ Subunits of Escherichia coli RNA Polymerase

(Received for publication, August 8, 1996, and in revised form, October 7, 1996)

Irina Artsimovitch ^¶ , Katsuhiko Murakami , Akira Ishihama and Martha M. Howe ^¶

From the ^¶ Department of Microbiology and Immunology, University of Tennessee-Memphis, Memphis, Tennessee 38163 and  Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, P_m, has a 10 hexamer but lacks a recognizable 35 hexamer. Interactions between Mor and RNA polymerase were studied using in vitro transcription, DNase I footprinting, and the yeast interaction trap system. We observed reduced promoter activity in vitro using reconstituted RNA polymerases with C-terminal deletions in  or ⁷⁰. As predicted if  were binding to P_m, we detected a polymerase-dependent footprint in the 60 region. Reconstituted RNA polymerases containing Ala substitutions in the  C-terminal domain were used to assay Mor-dependent transcription from P_m in vitro. The D258A substitution and  deletion gave large reductions in activation, whereas the L262A, R265A, and N268A substitutions caused smaller reductions. The interaction trap assay revealed weak interactions between Mor and both  and ⁷⁰; consistent with a key role of -D258, the D258A substitution abolished interaction, whereas the R265A substitution did not. We propose that: (i) -D258 is a Mor "contact site"; and (ii) residues Leu-262, Arg-265, and Asn-268 indirectly affect Mor-polymerase interaction by stabilizing the ternary complex via -DNA contact.

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