Transcription of bacteriophage T4 genome in vitro. Heterogeneity of RNA polymerase in crude extracts of normal and T4-infected Escherichia coli B

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Abstract
In order to obtain RNA polymerase preparations carrying the necessary specificity determinants to transcribe the delayed-early genes of bacteriophage T4, crude extracts of uninfected and T4-infected Escherichia coli were fractionated in glycerol gradients of low ionic strength. In contrast to the reported sedimentation behavior of the purified enzyme, the RNA polymerase activity in crude extracts of normal and infected cells sedimented heterogeneously over a wide range of sedimentation coefficients. When the "heavy" (24-33 S) and "light" (14-20 S) regions of the gradient were precipitated with ammonium sulfate and recentrifuged, the former split into two subfractions, one again sedimenting heavy and the other sedimenting light. The latter did not split under the same conditions. The resulting subfractions from uninfected cell extracts had different thermal thermal stabilities at 50 degrees (half-lives ranging from 2-3 to 25 min) while those from T4-infected cell extracts were very thermolabile (half-life of 1-2 min). All the subfractions were more active on T4 DNA than on calf-thymus DNA. They also formed rifampicin-resistant, RNA chain initiation complexes with T4 DNA. Based on the kinetics of heat inactivation with T4 and calf thymus DNAs as templates and preferential transcription of T4 DNA, it is proposed that the T4-infected cell enzymes prepared as described here harbor heat-labile initiation factor(s). During infection the heavy sedimenting RNA polymerase activity disappears after 2.5 min at 37 degrees. This appears to require phage-specific protein synthesis because (a) it does not happen in the presence of chloramphenicol and (b) it does not happen in T4 ghost-infected cells.

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