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Nucleic Acids Res. 2008 Nov;36(20):6396-405. doi: 10.1093/nar/gkn639. Epub 2008 Oct 5.

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

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  • 1Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

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 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.

PMID:
18838393
[PubMed - indexed for MEDLINE]
PMCID:
PMC2582627
Free PMC Article
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