Mutational analysis of beta '(260-309), a sigma(70) binding site located on Escherichia coli core RNA polymerase

eubacteria, the a subunit binds to the core RNA polymerase and directs tran scription initiation from any of its cognate set of promoters, Previously, our laboratory defined a region of the beta' subunit that interacts with si gma(70) in vitro. This region of beta' contained heptad repeat motifs indic ative of coiled coils. In this work, we used 10 single point mutations of t he predicted coiled coils, located within residues 260-309 of beta', to loo k at disruption of the sigma(70)-core interaction, Several of the mutants m ere defective for binding sigma(70) in vitro. Of these mutants, three (R275 Q, E295K, and A302D) caused cells to be inviable in an in vivo assay in whi ch the mutant beta' is the sole source of beta' subunit for the cell. All o f the mutants were able to assemble into the core enzyme; however, R275Q, E 295K, A302D were defective for E sigma(70) holoenzyme formation. Several of the mutants were also defective for holoenzyme assembly with various minor a factors. In the recently published crystal structure of Thermus aquaticu s core RNA polymerase (Zhang, G., Campbell, E. k, Minakhin, L,, Richter, C, , Severinov,,, and Darst, S, A. (1999) Cell 98, 811-824), the region homolo gous to beta'(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.