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Genetic analysis of phage Mu Mor protein amino acids involved in DNA minor groove binding and conformational changes
- Source :
- The Journal of biological chemistry. 286(41)
- Publication Year :
- 2011
-
Abstract
- Gene expression during lytic development of bacteriophage Mu occurs in three phases: early, middle, and late. Transcription from the middle promoter, P(m), requires the phage-encoded activator protein Mor and the bacterial RNA polymerase. The middle promoter has a -10 hexamer, but no -35 hexamer. Instead P(m) has a hyphenated inverted repeat that serves as the Mor binding site overlapping the position of the missing -35 element. Mor binds to this site as a dimer and activates transcription by recruiting RNA polymerase. The crystal structure of the His-Mor dimer revealed three structural elements: an N-terminal dimerization domain, a C-terminal helix-turn-helix DNA-binding domain, and a β-strand linker between the two domains. We predicted that the highly conserved residues in and flanking the β-strand would be essential for the conformational flexibility and DNA minor groove binding by Mor. To test this hypothesis, we carried out single codon-specific mutagenesis with degenerate oligonucleotides. The amino acid substitutions were identified by DNA sequencing. The mutant proteins were characterized for their overexpression, solubility, DNA binding, and transcription activation. This analysis revealed that the Gly-Gly motif formed by Gly-65 and Gly-66 and the β-strand side chain of Tyr-70 are crucial for DNA binding by His-tagged Mor. Mutant proteins with substitutions at Gly-74 retained partial activity. Treatment with the minor groove- and GC-specific chemical chromomycin A(3) demonstrated that chromomycin prevented His-Mor binding but could not disrupt a pre-formed His-Mor·DNA complex, consistent with the prediction that Mor interacts with the minor groove of the GC-rich spacer in the Mor binding site.
- Subjects :
- Mutation, Missense
Helix-turn-helix
Cell Cycle Proteins
Biology
Crystallography, X-Ray
Response Elements
Biochemistry
DNA-binding protein
Bacteriophage mu
mental disorders
polycyclic compounds
Drosophila Proteins
Gene Regulation
Binding site
Molecular Biology
Polymerase
Helix-Turn-Helix Motifs
Escherichia coli K12
Oligonucleotide
Chromomycin A3
Cell Biology
DNA Minor Groove Binding
Protein Structure, Tertiary
DNA binding site
nervous system
Amino Acid Substitution
DNA, Viral
biology.protein
human activities
Binding domain
Protein Binding
Subjects
Details
- ISSN :
- 1083351X
- Volume :
- 286
- Issue :
- 41
- Database :
- OpenAIRE
- Journal :
- The Journal of biological chemistry
- Accession number :
- edsair.doi.dedup.....29c54066bcc030c9913a7d9a142fa0db