RATE OF TRANSLOCATION OF BACTERIOPHAGE-T7 DNA ACROSS THE MEMBRANES OFESCHERICHIA-COLI

Translocation of bacteriophage T7 DNA from the capsid into the cell ha s been assayed by measuring the time after infection that each GATC si te on the phage genome is methylated by cells containing high levels o f DNA adenine methylase. Methylation at GATC sites on T7 DNA renders b oth the infecting genome and any newly synthesized molecules sensitive to the restriction enzyme DpnI. In a normal infection at 30 degrees C , translocation of the T7 genome into the cell takes between 9 and 12 min. In contrast, translocation of the entire phage lambda genome or o f a T7 gnome ejected from a lambda capsid can be detected within the f irst minute of infection, Entry of the leading end of the T7 genome oc curs by a transcription-independent mechanism that brings both Escheri chia coil and T7 promoters into the cell. Further translocation of the genome normally involves transcription by the RNA polymerases of both E. coli and T7; the rates of DNA translocation into the cell when cat alyzed by each enzyme are comparable to the estimated rates of transcr iption of the respective enzymes. A GATC site located between the earl y E. coli promoters and the coding sequences of the first T7 protein m ade after infection is not methylated before the protein is synthesize d, a result supporting the idea (B. A, Moffatt and F, W. Studier, J, B acteriol. 170:2095-2105, 1988) that only certain proteins are permitte d access to the entering T7 DNA. In the absence of transcription, the genomes of most T7 strains do not completely enter the cell, However, the entire genome of a mutant that lacks bp 393 to 808 of T7 DNA enter s the cell in a transcription-independent process at an average overal l rate of 50 bp per s.