Regional mutagenesis of the gene encoding the phage Mu late gene activator C identifies two separate regions important for DNA binding

Abstract

Lytic development of bacteriophage Mu is controlled by a regulatory cascade and involves three phases of transcription: early, middle and late. Late transcription requires the host RNA polymerase holoenzyme and a 16.5-kDa Mu-encoded activator protein C. Consistent with these requirements, the four late promoters Plys, PI, PP and Pmom have recognizable -10 hexamers but lack typical -35 hexamers. The C protein binds to a 16-bp imperfect dyad-symmetrical sequence element centered at -43.5 and overlapping the -35 region. Based on the crystal structure of the closely related Mor protein, the activator of Mu middle transcription, we predict that two regions of C are involved in DNA binding: a helix-turn-helix region and a β-strand region linking the dimerization and helix-turn-helix domains. To test this hypothesis, we carried out mutagenesis of the corresponding regions of the C gene by degenerate oligonucleotide-directed PCR and screened the resulting mutants for their ability to activate a Plys - galK fusion. Analysis of the mutant proteins by gel mobility shift, β-galactosidase and polyacrylamide gel electrophoresis assays identified a number of amino acid residues important for C DNA binding in both regions. © 2008 The Author(s).