To determine the direction of the entry of DNA during in vitro bacteriophage T7 DNA packaging, incompletely packaged DNA (ipDNA) was fractionated by agarose gel electrophoresis after degradation of DNA outside of capsids and then release of packaged DNA from capsids. After fractionation, quantitative in-gel probing with a right end-specific oligonucleotide detects heterogeneous ipDNA (called right-end ipDNA). Most of the right-end ipDNA appears with kinetics expected of a precursor to the mature T7 DNA. In-gel probing with a left-end-specific oligonucleotide detects ipDNA (left-end ipDNA); the molar amount of left end ipDNA is always at least 50× less than the molar amount of right-end ipDNA. Left-end ipDNA appears with the kinetics of an abortive end product of T7 DNA packaging. Thus, productive T7 DNA packaging occurs in a right-to-left direction. Quantitation of the conversion of right-end ipDNA to mature-length DNA yields an estimate of the mean rate of right-to-left in vitro T7 DNA packaging: 28 ± 6 kbp/min for the last 20-50% of the DNA packaged.
ASJC Scopus subject areas