Utilization of bacteriophage T7 late promoters in recombinant plasmids during infection

Abstract

When bacteriophage T7 infects a cell that carries a recombinant plasmid having a promoter for T7 RNA polymerase, the cloned promoter is utilized in the plasmid. A simple hybridization test has been used to screen recombinant plasmids for T7 promoter activity in vivo and to analyze the kinetics of utilization of the promoters in the plasmids. At least 13 different T7 promoters have been found to be active in plasmids, and all can be correlated with promoters identified by other means. During infection, promoters in T7 DNA appear to be activated sequentially from left to right over a period of several minutes; however, regardless of their original position in the T7 DNA molecule, T7 promoters in plasmids are all activated at the same time as the first promoters in T7 DNA. A likely explanation for this difference is that T7 DNA enters the cell in stages, and that not all promoters are accessible at the time T7 RNA polymerase is first made; promoters in plasmids, on the other hand, would be immediately accessible. Phased entry of the T7 DNA molecule could also explain why class II promoters are utilized before the stronger class III promoters during T7 infection. Utilization of a promoter in a plasmid seems to shut off with the same kinetics as the equivalent promoter in T7 DNA, and a mutation in T7 lysozyme that causes prolonged utilization of class II promoters in T7 DNA has a similar effect on class II promoters in plasmids. Transcription of promoter-containing plasmid DNAs by purified T7 RNA polymerase terminates at specific sites in pBR322 DNA, but the efficiency of termination is not high, and RNAs that result from transcription several times around the plasmid DNA are made in both directions. The ability to utilize T7 promoters in plasmids suggests that this system could be developed for high efficiency of expression of cloned genes in bacteria.