Identification of the domains for DNA binding and transactivation function of C protein from bacteriophage Mu.
The C protein, a middle gene product of bacteriophage Mu, is the determinant of the transition from middle to late gene expression. C activates transcription from four late gene promoters, P(lys), P(I), P(P), and P(mom) by binding to a site overlapping their -35 elements. Site-specific, high-affinity binding of C to its recognition sequence results in both axial and torsional distortion of DNA at P(mom), which appears to play a role in recruitment of RNA polymerase to the promoter for mom gene transactivation. To identify the regions of C protein important for its function, deletion and site-directed mutagenesis were carried out. We demonstrate here that a helix-turn-helix (HTH) motif located toward the carboxy terminal end of the protein is the DNA-binding domain and amino acid residues involved in transactivation overlap the HTH motif. Mutagenesis studies also aided in the identification of the region important for dimerization. Structure-based sequence alignment and molecular modeling in conjunction with mutational analysis suggest that the HTH motif is part of a three-helix bundle, with remarkable similarity to paired (prd), a developmental regulatory protein from Drosophila. Additional key residues identified in the model to be crucial for C protein structure and DNA binding were shown to be important by mutagenesis. These results provide a structural framework for C function and insight into the mechanism of transactivation at the mom promoter.
Paul
Bindu Diana
BD
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-560012, India.
Kanhere
Aditi
A
Chakraborty
Atanu
A
Bansal
Manju
M
Nagaraja
Valakunja
V
D000595:Amino Acid Sequence; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D019077:Helix-Turn-Helix Motifs; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014764:Viral Proteins
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C090591:C protein, Bacteriophage Mu; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014764:Viral Proteins