Entry of bacteriophage T7 DNA into the cell and escape from host restriction

Abstract

T7 DNA did not become susceptible to degradation by the host restriction enzymes EcoB, EcoK, or EcoP1 until 6 to 7 min after infection (at 30 degrees C). During this period, T7 gene 0.3 protein is made and inactivates EcoB and EcoK, allowing wild-type T7, or even a mutant that has recognition sites flanking gene 0.3, to escape restriction by these enzymes. However, T7 failed to escape restriction by EcoP1 even though 0.3 protein was made, evidently because 0.3 protein is unable to inactivate EcoP1. How T7 DNA can be accessible to transcription but not restriction in the first few minutes of infection is not yet understood, but we favor the idea that the entering DNA is initially segregated in a special place. Entry of T7 DNA into the cell is normally coupled to transcription. Tests of degradation of DNAs having their first restriction sites different distances from the end of the DNA indicated that only the first 1,000 or so base pairs (2.5%) of the molecule enter the cell without transcription. An exception was the only mutant tested that lacks base pairs 343 to 393 of T7 DNA; most or all of this DNA entered the cell without being transcribed, apparently because it lacks a sequence that normally arrests entry. This block to DNA entry would normally be relieved by the host RNA polymerase transcribing from an appropriately situated promoter, but the block can also be relieved by T7 RNA polymerase, if supplied by the host cell. T7 mutants that lack all three strong early promoters A1, A2, and A3 could grow by using a secondary promoter.