Interaction of the bacteriophage Mu transcriptional activator protein, C, with its target site in the mom promoter

[摘要] 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, P-mom, P-lys, P-I and P-P. 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 P-mom. The results indicate that C contacts P-mom 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-P-mom 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 P-mom DNA. (C) 1997 Academic Press Limited.

[发布日期] 1997-11-07

[关键词] phage Mu late promoters;C protein-DNA interaction;transcriptional activation;chemical protection and interference analysis;mutagenesis