Analysis of the fine structure of the prohead binding domain of the packaging protein of bacteriophage T3 using a hexapeptide, an analog of a prohead binding site.
(15/25)
A large subunit of bacteriophage T3 packaging enzyme, a product of gene 19 (gp19, 586 amino acid residues), binds a prohead prior to DNA translocation in DNA packaging. Its C-terminal region (571 to 576, Region I) is of crucial importance for prohead binding. To elucidate the functional role(s) of Region I in DNA packaging, a hexapeptide (6pT3) corresponding to the Region I sequence and its variants were synthesized and their effects on DNA packaging in a defined in vitro system were examined. 6pT3 did not inhibit gp19wt (wild type)-prohead binding but interfered with their functional interaction, resulting in inhibition of DNA packaging. The inhibitory effect of 6pT3 on gp19wt was reversible. The effect of 6pT3 was examined with gp19 delta C10, which was active in DNA packaging in spite of lacking the extreme C-terminal 10 amino acids (Region II). The inhibitory effect on gp19 delta C10 was more severe than that on gp19wt and was irreversible. From these results, we concluded that the prohead binding domain is composed of two subdomains: Region I is a "core" domain, and its binding to the prohead is crucial for DNA packaging, and Region II is an "anchor" domain stabilizing the binding by Region I. (+info)
A novel method for generating nested deletions using the in vitro bacteriophage T3 DNA packaging system.
(16/25)
To sequence a DNA segment inserted into a cosmid vector under the directed sequencing strategy, we established a simple and rapid method for generating nested deletions which uses the in vitro packaging system of bacteriophage T3 DNA. The principle is based on the previous finding that this system can translocate any linear double-stranded DNA up to 40 kb into the phage capsid in a time-dependent manner and the encapsulated DNA becomes DNase-resistant. For this purpose, we constructed a cosmid vector that carries two different antibiotic selection markers at both sides of the multiple cloning site, and after insertion of a DNA segment, the clone was linearized by lambda-terminase at the cos site. After the packaging reaction in vitro followed by DNase treatment, the encapsulated DNA was introduced into Escherichia coli cells to give clones with unidirectional deletions by differential antibiotic selection. Restriction and sequence analyses of deletion clones demonstrated that an ordered set of clones with nested deletions, ranging from less than 1 kb to 25 kb, was created from either the end of the DNA segment. Thus, nested deletion clones that cover the entire region of a approximately 40-kb cosmid insert can be obtained by a single packaging reaction, and its restriction map can be simultaneously obtained. (+info)