Nucleic Acids Research Advance Access published online on October 5, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn639
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Molecular Biology |
Regional mutagenesis of the gene encoding the phage Mu late gene activator C identifies two separate regions important for DNA binding
Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, USA
*To whom correspondence should be addressed. Tel: +1 901 448 8215; Fax: +1 901 448 8462; Email: mhowe{at}utmem.edu
Received June 29, 2008. Revised August 29, 2008. Accepted September 16, 2008.
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.
Present address: Yide Jiang, Oncology, Genzyme Corporation, 5 Mountain Road, Framingham MA 01701-9322