Construction and Characterization of a Plasmid Borne Prokaryotic-Eukaryotic Fusion RNA Polymerase Largest Subunit
Rieden, Cynthia L.
A novel plasmid, pKC91, was created. The plasmid encodes the Escherichia coli RNA polymerase largest subunit β rpoC gene with an altered carboxyl-terminal region. The C-terminal region was modified to include the heptapeptide repeat of Saccharomyces cerevisiae RNA polymerase largest subunit. The repeat has the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser, and has been implicated in the processes of DNA binding and RNA chain elongation. The addition of this region was found to have no effect on growth rate or protein expression of transfected cells. It did interfere with functional complementation in rpoC deficient strains. The addition of the heptapeptide repeat disrupts function of the produced RNA polymerase largest subunit, potentially by steric hinderance of the catalytic site, inhibition of the ability to complex with other subunits to form the holoenzyme, or by replacement of a region essential for function.
iv, 33 p.
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