Regional mutagenesis of the gene encoding the phage Mu late gene activator C identifies two separate regions important for DNA binding.
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 P(lys), P(I), P(P) and P(mom) 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 beta-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 P(lys)-galK fusion. Analysis of the mutant proteins by gel mobility shift, beta-galactosidase and polyacrylamide gel electrophoresis assays identified a number of amino acid residues important for C DNA binding in both regions.
Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Yide
Y
Jiang
Martha M
MM
Howe
D000595:Amino Acid Sequence; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C090591:C protein, Bacteriophage Mu; D014764:Viral Proteins; D004247:DNA