Interaction of the bacteriophage Mu transcriptional activator protein, C, with its target site in the mom promoter.
The bacteriophage Mu C gene encodes a 16.5 kDa site-specific DNA binding protein that is a transcriptional activator of the four "late" promoters, Pmom, Plys, PI and PP. A symmetrical consensus C recognition sequence, TTAT[N5-6]ATAA, containing an inverted tetrad repeat separated by a spacer of five to six G+C-rich nucleotides, has been proposed. To investigate this, we used oligonucleotide mutagenesis to introduce random substitutions within and flanking the proposed C-target region; each variant site was tested for C recognition by an in vivo functional transactivation assay. We observed that all single mutations, in either tetrad, reduced C activation. Although two out of ten substitutions within the spacer reduced activation, the spacer region does not appear to make specific contact with C. We also used in vitro chemical-protection and -interference to study C contacts with Pmom. The results indicate that C contacts Pmom DNA on only one face of the helix through interactions within two adjacent major grooves; this conclusion was supported by gel shift analyses using synthetic oligonucleotide duplexes containing I.C or other base-pair substitutions. Evidence is also presented that C-Pmom contacts are asymmetrical, and that they extend two nucleotides 3' to the promoter-proximal tetrad. We also show that C binding induces a deformation, possibly a bend, in Pmom DNA.
Sun
W
W
Department of Biology, University of Rochester, NY 14627, USA.
Hattman
S
S
Kool
E
E
D010583:Bacteriophage mu / Q000235:genetics*; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites / Q000235:genetics; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C090591:C protein, Bacteriophage Mu; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014764:Viral Proteins