title: Genetic analysis of phage Mu Mor protein amino acids involved in DNA minor groove binding and conformational changes.
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.
authors.affiliation: Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
authors.forename: Muthiah
authors.initials: M
authors.lastname: Kumaraswami
authors.affiliation: Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Avanigadda
authors.affiliation: Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Rai
authors.affiliation: Department of Pharmacology, Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G1L7 Canada.
authors.forename: Hee-Won
authors.initials: HW
authors.lastname: Park
authors.affiliation: Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163. Electronic address: mhowe@uthsc.edu.
authors.forename: Martha M
authors.initials: MM
authors.lastname: Howe
mesh_terms: D019943:Amino Acid Substitution; D010583:Bacteriophage mu / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014128:Chromomycin A3 / Q000737:chemistry; D018360:Crystallography, X-Ray; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D048168:Escherichia coli K12 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000821:virology; D019077:Helix-Turn-Helix Motifs; D020125:Mutation, Missense; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020218:Response Elements
chemical_list: D018797:Cell Cycle Proteins; D004279:DNA, Viral; D029721:Drosophila Proteins; C462943:mor protein, Drosophila; D014128:Chromomycin A3