Bacteriophage T7 is the paradigm for studying DNA translocation across cell membranes. A powerful assay, that quantitates rates of genome transport from the phage virion into the cell, allows dissection of the underlying in vivo mechanisms. The assay also detects changes in the in vivo rates of transcription following antitermination; it will be used to understand transcription antitermination in vivo using T7 and phage HK022. The long-term goal of this work is to provide a complete mechanistic description of the initial steps of viral infection, including DNA transport across the cell envelope and into the cytoplasm. These studies have broad implications for the mechanisms and energetics of nucleic acid transport across hydrophobic lipid bilayers in all living cells. The combination of a powerful assay and established genetic systems for both the virus and its host make this goal achievable. T7 proteins, ejected from the virion into the cell, not only form a channel across the cell envelope used for DNA translocation but also reconstitute an enzyme that ratchets the 40 kb genome into the cell at approximately 70 bp/sec. Existing gene 16 mutants, known to be defective in DNA translocation, will allow the intracellular location of the ejected proteins to be determined, and the transmembrane domains of gp16 to be defined and chemically probed. The role of the membrane potential in channel formation and maintenance will be evaluated. Additional mutants will be isolated that will help define the active site for DNA translocation in gp16 and to determine the physiological function of the gene 15 protein in the initiation of infection. Normally, transcription internalizes most of the T7 genome. The role of each early promoter in genome entry and the physiological significance of the T7 antitermination system will be characterized in vivo and in vitro. The interference of the A3 promoter and its antitermination element in T7 DNA packaging, and the role of T7 gp2 in overcoming interference will be elucidated using in vivo and in vitro approaches.
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