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J. Biol. Chem., Vol. 275, Issue 30, 23113-23119, July 28, 2000

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Mutational Analysis of '_260-309, a ⁷⁰ Binding Site Located on Escherichia coli Core RNA Polymerase^*

Terrance M. Arthur^§, Larry C. Anthony^§, and Richard R. Burgess^¶

From the  McArdle Laboratory for Cancer Research and the ^§ Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

In eubacteria, the  subunit binds to the core RNA polymerase and directs transcription initiation from any of its cognate set of promoters. Previously, our laboratory defined a region of the ' subunit that interacts with ⁷⁰ in vitro. This region of ' contained heptad repeat motifs indicative of coiled coils. In this work, we used 10 single point mutations of the predicted coiled coils, located within residues 260-309 of ', to look at disruption of the ⁷⁰-core interaction. Several of the mutants were defective for binding ⁷⁰ in vitro. Of these mutants, three (R275Q, E295K, and A302D) caused cells to be inviable in an in vivo assay in which the mutant ' is the sole source of ' subunit for the cell. All of the mutants were able to assemble into the core enzyme; however, R275Q, E295K, A302D were defective for E⁷⁰ holoenzyme formation. Several of the mutants were also defective for holoenzyme assembly with various minor  factors. In the recently published crystal structure of Thermus aquaticus core RNA polymerase (Zhang, G., Campbell, E. A., Minakhin, L., Richter, C., Severinov, K., and Darst, S. A. (1999) Cell 98, 811-824), the region homologous to '_260-309 of Escherichia coli forms a coiled coil. Modeling of our mutations onto that coiled coil places the most defective mutations on one face of the coiled coil.

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