Characterization of bacteriophage T7 DNA entry into Escherichia coli

n assay has been developed that measures the rate of translocation of T7 DNA into the cell; this assay has allowed the process of DNA entry to be dissected. Analysis of the kinetics of entry of several deletion mutants, constructed in vitro as part of this study, shows that less than 877 base-pairs (bp) of the 40 kilobase pair (kb) chromosome is ejected from the viral particle into the cytoplasm. Promoters on this 877 bp allow immediate transcription by Escherichia coli RNA polymerase. As the transcribing polymerase tracks along the partially ejected genome, DNA enters the cell at a rate of 40-50 bp per sec, a rate corresponding to that of E. coli mRNA synthesis. Further DNA translocation is catalyzed by T7 RNA polymerase at 250 bp per sec, a rate also corresponding to that enzyme's rate of transcription in vivo. Therefore, RNA polymerases pull the T7 chromosome into the cell as they transcribe.;In the absence of transcription, the T7 chromosome partially penetrates the cell. Between 9 kb and 30 kb of DNA enter the cytoplasm by an inefficient, highly temperature-dependent mechanism. However, a genome that contains an ectopically placed EcoK recognition site near the leading end is efficiently translocated by the restriction enzyme EcoK; T7 DNA translocation is therefore not strictly dependent on transcription. Mutants altered in gene 16 have been isolated and characterized; the mutations permit complete genome translocation in the absence of transcription. Thus gp16, an internal virion protein, is probably involved in retarding transcription- independent translocation after virion-catalyzed ejection.;T7 DNA ejection from a...