The cloning and characterization of the bacteriophage D108 regulatory DNA-binding protein ner

From the transposable Mu-like bacteriophage D108 we have cloned the ner gene under the control of the lac UV5 promoter in the expression vector pOP95-15. The recombinant plasmid, pPT011, overproduced the 8-kd D108 ner protein (visualized by in vitro-coupled transcription-translation) and served as a substrate for DNA sequencing of the D108 ner gene. The ner protein of D108 was found to be 48% homologous to the Mu ner protein, though the DNA sequences that encode these proteins are quite divergent. We used the retardation of migration of 32P-labelled DNA restriction fragments by ner-containing crude protein extracts in polyacrylamide gels (band competition assay) to determine which DNA restriction fragment(s) contained the ner-binding sites. DNA footprinting using crude extracts physically identified the 47-bp DNA sequence that the ner protein was interacting with in the D108 early gene regulatory region. This sequence is located 10 bp downstream from the presumed D108 early gene transcription initiation site. Therefore, by binding strongly to this 47-bp DNA sequence, the D108 ner protein can regulate D108 early gene transcription.