Amplification and isolation of Escherichia coli nusA protein and studies of its effects on in vitro RNA chain elongation.
The Escherichia 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, we have cloned the nusA gene into a lambda PL-derived overexpression vector. E. coli strains bearing the resulting plasmid (pMS7) produce large amounts of nusA protein when induced, and the protein is easily purified to homogeneity. Biochemical studies of nusA protein reveal that it inhibits in vitro RNA chain elongation by E. coli RNA polymerase with a variety of templates. Two modes of inhibition are found. Inhibition of elongation with poly[d(A-T] ) template is completely competitive with nucleoside triphosphates and shows an inhibitory constant (Ki) of 3 X 10(-7) M. In contrast, inhibition of elongation with T7 DNA as template is mixed. One component of the inhibition is competitive with nucleoside triphosphate substrates and is reversed at elevated substrate concentrations. A second inhibitory component remains even at saturating substrate concentrations; this sequence-dependent mode of inhibition shows a much lower Ki of 2 X 10(-8) M. The existence of two different modes of inhibition might be explained if two molecules of nusA protein can bind to each RNA polymerase complex. The interaction of nusA protein with elongating RNA polymerase molecules is not processive but appears to be characterized by rapid association and dissociation. Under proper conditions, a sigma-nusA cycle [Greenblatt, J., & Li, J. (1981) Cell (Cambridge, Mass.) 24, 421-428] can be demonstrated in vitro in which each polymerase goes through multiple rounds of transcription involving successive interactions with sigma and the nusA protein.
D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification; D005796:Genes; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007700:Kinetics; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes