Structure-function analysis of the bacteriophage PRD1 DNA terminal protein: Nucleotide sequence, overexpression, and site-directed mutagenesis of the terminal protein gene.
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PublisherThe University of Arizona.
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AbstractThe nucleotide sequence of the PRD1 terminal protein gene has been determined. The coding region for PRD1 terminal protein is 777 base pairs long and encodes 259 amino acid residues (29,326 daltons). The deduced amino acid sequence of PRD1 terminal protein reveals no overall homology with other known terminal proteins or related proteins. A closer examination revealed a highly conserved amino acid sequence, YSRLRT, exist among all identified DNA terminal proteins including PRD1, PZA, Nf, φ29 and adenovirus. This is the first conserved amino acid sequence that has been found in all identified DNA terminal proteins. Not only is the YSRLRT sequence conserved, but its spatial location is similar as well. Therefore, the significance of the YSRLRT conserved sequence is suggested by both its conservative spatial location and high degree of homology across species. To study the structure-function relationship of the YSRLRT sequence of PRD1 terminal protein, in vitro site-directed mutagenesis was performed to determine the role of each amino acid in this conserved region. The PRD1 terminal protein and DNA polymerase genes were cloned into phagemid pEMBLex3, and the recombinant plasmid used for constructing mutants. Eleven PRD1 terminal protein mutant clones were examined for their priming complex formation activities. Our results have strongly demonstrated that the positive charge residue of arginine-174 plays an important role for PRD1 terminal protein function. There are 13 tyrosine residues in the predicted PRD1 terminal protein. It was of interest to known which tyrosine is actually linked to terminal nucleotide of the PRD1 DNA. We used a new approach involving replacing the tyrosine residues with phenylalanine residues in the carboxyl terminal portion of the protein. From analyses, the tyrosine-190 has been determined to be the most likely linkage site between terminal protein and PRD1 DNA.
Degree ProgramMicrobiology and Immunology