Bacteriophage T7 has become a paradigm for studying DNA translocation across cell membranes. Proteins ejected from the virion into the cell form a molecular motor that ratchets the phage genome into the cell at about 70 bp/sec at 30oC, using the proton motive force as its source of energy. Normally only 850 bp of the 40 kb genome are ejected by this motor, but mutants were characterized that translocate the entire genome into the cell by this mechanism. Most of the phage genome is brought into the cell via transcription, and the established assay for DNA translocation will be used to measure kinetic parameters of several DNA translocating enzymes in vivo. The combination of this powerful assay and the established genetic systems available for both T7 and its host make the complete elucidation of the mechanism by which a phage genome enters the cell a probable result of this research. The data that will be obtained have broad implications in a general understanding of the mechanisms and energetics of nucleic acid translocation across hydrophobic lipid bilayers in all biological systems. Phage mutants that are defective in the virion motor have been isolated and will be used to characterize the initial steps of the infection process, including establishment of the transmembrane channel in vivo. The proteins involved will be purified, the membrane-insertion, DNA-binding, and potential transglycosylase activities of gpl6 will be tested in vitro; these properties are strongly indicated from previous in vivo studies. The proposed experiments form the initial steps towards attempting to reconstruct the virion motor in vitro. Phage mutants that affect steps at or near the initiation of infection have been, and will continue to be, isolated. Their analysis is designed towards an understanding of the signal transduction pathway from the cell surface into the phage head that triggers protein, and then DNA, ejection from the virion.
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