DNA sequences necessary for packaging bacteriophage T3 DNA

Abstract

A recombinant plasmid, pUCE1-TR, carrying a target for processing of the concatemer joint (TR) and sequences to the left of the target (E1), is efficiently packaged into transducing particles during T3 phage infection. Using this plasmid packaging/transduction system, the minimal sequences necessary for packaging of T3 DNA were determined. The TR sequence contains the targets for initiation cleavage and termination cleavage of concatemer processing ( pacCR and pacCL, respectively). A plasmid lacking pacCL was packaged as efficiently as pUCE1-TR but one deleted for pacCR was packaged at a very low efficiency, showing that pacCR is essential for production of transducers but that pacCL is dispensable. DNA from transducing particles carrying a recombinant plasmid lacking pacCL or pacCR had the same right or left end as T3 DNA, respectively, but its other end was not unique. In the absence of pacCL, packaging is initiated from the DNA end created by cleavage at the pacCR and terminated at any sequence after packaging a headful of DNA. In the absence of pacCR, packaging is initiated from the DNA end created by nonspecific, inefficient cleavage and terminated by cleavage at the pacCL after packaging a headful of DNA. A 23-bp segment flanking the site where the mature right end is formed was found to support efficient formation of transducing particles. A 53-bp sequence, including a consensus sequence for the promoter for T3 RNA polymerase, was a responsible element in the El sequence for packaging of plasmid DNA. Deletions of the 5′-upstream sequence of the promoter sequence from the left decreased the promoter and packaging activities in parallel, but with those of the 3′-downstream sequence from the right, the packaging activity was impaired before the promoter activity, indicating that transcription from the promoter is necessary but not sufficient for T3 DNA packaging.