Identification and purification of the N gene product of bacteriophage phi 80

To confirm the in vivo observation that the N gene product of phi 80, phi 80-pN, prevents termination of transcription at the tL1 region and is therefore a transcription antitermination factor (Tanaka and Matsushiro 1985), we demonstrated that phi 80-tL1 is a rho-dependent terminator, similar to lambda-tL1, and that phi 80-pN has a transcription antitermination function at this site in an in vitro transcription system using a nucleic acid-free S-100 extract. In the presence of rho-protein, transcription termination at tL1 was suppressed completely with an S-100 extract prepared from Escherichia coli strain NT525 containing the pBN1-N+ plasmid. Starting from this pN-overproducing cell extract, we purified phi 80-pN to homogeneity by chromatography on DEAE-Sephacel, Sephadex G-150 and CM-Sephadex C-50. The molecular weight of purified pN was about 12,000 and the NH2-terminal sequence was NH2-Met-Ile-Asp-Asp-Ile-Lys, which was consistent with the sequence deduced from the DNA sequence.