The "host shutoff' function of bacteriophage T7 involves an inactivation of the host Escherichia coli RNA polymerase by an inhibitor protein bound to the enzyme. When this inhibitor protein, termed I protein, was removed from the inactive RNA polymerase complex prepared from T7-infected cells by glycerol gradient centrifugation in the presence of 1 M KCl, the enzyme recovered its activity equivalent to about 70 to 80% of the activity of the enzyme from uninfected cells. Analysis of the activity of E. coli RNA polymerase from E. coli cells infected with various T7 mutant phages indicated that the T7 gene 2 codes for the inhibitor I protein. The activity of E. coli RNA polymerase from gene 2 mutant phage-infected cells, which was about 70% of that from uninfected cells, did not increase after glycerol gradient centrifugation in the presence of 1 M KCl, indicating that the salt-removable inhibitor was not present with the enzyme. which indicated that gene 2 of T7 codes for the I protein. We also present evidence that, in addition to gene 2, T7 gene 0.7 which codes for a protein kinase (12, 22) is also required in accomplishing complete shutoff of the host E. coli RNA polymerase activity in T7 infection. MATERIALS AND METHODS Chemicals. [5-3H]UTP(22.7 Ci/mmol

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Bacteriophage T7-coded inhibitor ofEscherichia coli RNA polymerase, termed I protein, was purified from an inactive E. coli RNA polymerase-I protein complex isolated from phage T7-infected cells. A molecular weight of about 7,000 to 9,000 was assigned to the purified I protein by acrylamide-sodium dodecyl sulfate gel electrophoresis, Sephadex G-50 gel filtration, and glycerol gradient centrifugation analysis. I protein inhibits initiation of RNA synthesis by directly binding to the RNA polymerase holoenzyme and prevents the binding of the enzyme to the promoter sites on the template T7 DNA. However, once a highly stable transcriptional preinitiation complex between RNA polymerase holoenzyme and T7 DNA is formed at the promoter site on T7 DNA in the absence of MATERIALS AND MhEHODS Chemicals. [5-3H]UTP (22.7 Ci/mmol), [G-3H]leucine (40 Ci/mmol), and [methyl-3H]thymidine (6.7 Ci/ mmol) were purchased from New England Nuclear Corp.; ATP, CTP, GTP, and UTP were from Sigma Chemical Co.

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