In vitro transcription of phage T4 late genes on purified DNA by partially purified RNA polymerase from T4-infected Escherichia coli B cells

Abstract

RNA polymerase was purified from ‘late’ phage T4-infected Escherichia coli B cells by DNA-cellulose affinity chromatography and high salt agarose filtration. The DNA-cellulose-purified RNA polymerase preparation contained T4-coded DNA endonuclease activity and several proteins, some with sizes comparable with the known T4 maturation factors, essential for late RNA synthesis. Some of these proteins, and the DNA endonuclease utilizing native, parental T4 DNA and supercoiled ΦX 174 DNA as substrates, were partially separated from the RNA polymerase as a complex during agarose filtration. In vitro RNA was made by the DNA-cellulose-purified RNA polymerase using native, parental T4 DNA as template. About 26% of the in vitro RNA was transcribed from the DNA r-strand; 75% from the same r-strand region as in vivo late after infection. Both the abundancy and specificity of the in vitro r-strand transcription were markedly reduced after agarose filtration of the enzyme. Addition of the proteins separated from the RNA polymerase during agarose filtration caused a restoration of in vitro r-strand transcription abundancy, but not its specificity. These results show that partially purified RNA polymerase from T4-infected E. coli B cells was able to transcribe late T4 genes in vitro with some abundancy and specificity on purified, parental T4 DNA, but further purification of the enzyme caused an irreversible reduction of this ability.