The overproduction and characterization of the bacteriophage Mu regulatory DNA-binding protein ner

Abstract

The bacteriophage Mu ner gene has been cloned under the control of the lac UV5 promoter in the expression vector pOP95-15. The gene products of the recombinant plasmid, pUD88, visualized by in vitro coupled transcription-translation, are the bacteriophage Mu ner protein (8 kDa) and a 23 kDa protein consisting of the amino terminus of gpA (Mu transposase) fused to the carboxy terminus of β-lactamase. DNA-binding activity was measured by the retardation of migration of a 32P-labeled DNA restriction fragment (containing the presumed ner-binding sites) in polyacrylamide gels. We have demonstrated specific association of ner to its binding sites to occur within 30 sec after the addition of impure extracts of ner overproducing cells. Much of this binding was dissociated within 30 sec by competition with a 20-fold molar excess of specific unlabeled DNA restriction fragment, but was resistant to dissociation when competed with unlabeled heterologous DNA for as long as 45 min at 37°. By adapting a method for DNA-footprinting using impure extracts of ner overproducing cells. we were able to determine that the ner-binding sites are located between nucleotides 1026 and 1058 from the Mu left end. These results support the hypothesis that ner is similar to the cro regulatory protein from bacteriophage λ and acts to regulate Mu early gene expression and the choice between lytic and lysogenic development.