Abstract
Middle-mode RNA synthesis in T4-infected cells takes place before replication of phage DNA commences. What distinguishes it from early-mode RNA synthesis is that initiation of middle RNA depends on T4-coded proteins, in particular on the mot gene product. mot protein is localized in a DNA-protein complex which forms during the first few minutes of infection. All of the cell's mot protein is bound in this complex, and it continues to be bound long after the synthesis of mot protein has stopped. When we infect Escherichia coli with T4 carrying a temperature-sensitive mutation in the mot gene, we find a correlation between the physiology of this mot mutant and the amount of mot protein bound in the DNA-protein complex. Although there is some host RNA polymerase in the complex, mot protein does not seem to bind to this enzyme. Two other T4-coded proteins, of molecular weights 17,600 and 15,000, are also found in the pre-replicative DNA-protein complex. One of these, p17,600, is coded for by a 750-base pair region located between genes 39 and 56; p17,600 appears to be the recently described motB gene product. The other protein, p15,000, is not an RNA polymerase-binding protein; it is characterized by its strong binding to the DNA-protein complex.
Highlights
Middle-modeRNA synthesis in T4-infectedcells takes place before replication of phage DNAcommences
We have previously shown that mot protein synthesis deis still in large pieces
The T4 tsmGo1t gene productis temperature sensitive, After Sepharose2R filtration, we find the DNA-protein combut its behavior even at the permissive temperature of 30 "C plex enriched in the fsG1 mot protein
Summary
~ ~ A - P ~ oCot me p~lexnes-Manoil et al (14) have published a procedure for the isolation of nucleoprotein complexes containing replicating T 4 DNA. If a similar procedure is applied to cells in which T4 hasdeveloped for only 5 min at 30 “C (cf “Experimental Procedures”), pre-replicativeT4 DNA-protein complexes can be isolated in the excluded volume after Sepharose 2B column chromatography (Fig. 1A). We found that these complexes contain 4-5% of the [35S]methioninepulselabeled proteins layered onto the column. 0.8% of the labeled E. coli proteins applied to a Sepharose 2B column is found in the excluded volume This is the same percentage that had been found by Manoil et al (14) for complexes between replicating T4 DNA and host proteins. Used a T4 straincarrying a ts mutation itnhe mot gene, tsG1
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