Bacteriophage HK022 Nun Protein: A Specific Transcription Termination Factor that Excludes Bacteriophage λ

Abstract

Publisher Summary This chapter discusses the bacteriophage HK022 nun protein. Prophage HK022 excludes superinfecting phage λ by terminating transcription on the lambda chromosome. This exclusion is promoted by the 109 amino acid HK022 Nun protein. Nun carries an arginine-rich RNAbinding motif (ARM) at its N terminus. The chapter illustrates the procedure for purification of Nun which is explained in two parts—namely, induction and purification. The Nun overproducer plasmid pT7NunII is a pET-21d (+) derivative that carries nun between the plasmid NcoI and HindIII sites. The nun gene is under the control of the T7 promoter, which is in turn, is controlled by an adjacent lac operator and a lacI gene. Basal Nun expression is highly down-regulated by the T7 lysozyme encoded by the pLysS plasmid. E. coli carrying the pT7NunII plasmid is maintained in LB–ampicillin. The purification step includes soluble extract, SP-sepharose FF column chromatography, and mono S chromatography. The chapter includes the protocol for assaying of Nun proteins. Most of the Nun protein in the induced culture is soluble, and it is readily purified from a supernatant fraction by two chromatographic steps. In vitro transcription is performed with template derived from a HindIII-digested λ genome that contains the pL–nutL segment.