Amplification and isolation of E. coli nusA protein and studies of its effects on in vitro RNA chain elongation

Abstract

The E. coli nusA gene product is an RNA polymerase binding protein which has been implicated in a variety of cellular and viral termination and antitermination processes. To facilitate large-scale preparation and biochemical studies of the nusA protein, the nusA gene was cloned into a .lambda. PL-derived overexpression vector. E. coli strains bearing the resulting plasmid (pMS7) produced large amounts of nusA protein when induced, and the protein was easily purified to homogeneity. Biochemical studies of nusA protein revealed that it inhibits in vitro RNA chain elongation by E. coli RNA polymerase with a variety of templates. Two modes of inhibition were found. Inhibition of elongation with poly[d(A-T)] template was completely competitive with nucleoside triphosphates and showed an inhibitory constant (Ki) of 3 .times. 10-7 M. Inhibition of elongation with T7 DNA as template was mixed. One component of the inhibition was competitive with nucleoside triphosphate substrates and was reversed at elevated substrate concentrations. A 2nd inhibitory component remained even at saturating substrate concentrations; this sequence-dependent mode of inhibition showed a much lower Ki of 2 .times. 10-8 M. The existence of 2 different modes of inhibition might be explained if 2 molecules of nusA protein can bind to each RNA polymerase complex. The interaction of nusA protein with elongating RNA polymerase molecules was characterized by rapid association and dissociation. Under proper conditions, a .sigma.-nusA cycle could be demonstrated in vitro in which each polymerase goes through multiple rounds of transcription involving successive interactions with .sigma. and the nusA protein.