Construction and properties of a cell-free system for bacteriophage T4 late RNA synthesis

A cell-free system for synthesizing bacteriophage T4 late RNA is described. The system, which is based on the "cellophane disc" technique introduced by Schaller and co-workers (Schaller, H., Otto, B., Nüsslein, V., Huf, J., Hermann, R., and Bonnhoeffer, F. (1972) J. Mol. Biol. 63, 183-200), provides favorable conditions for T4 DNA and RNA synthesis in vitro. Total RNA synthesis can be sustained for more than 1 h at 25 degrees C and initiation of early and late RNA chains occurs in vitro. The capacity to yield cell-free systems which make T4 late RNA in vitro is acquired by virus-infected cells as they make late RNA in vivo. The in vitro synthesized RNA is highly asymmetric. The conditions which optimize the in vitro system with respect to several parameters (total extent of T4 transcription, rate of transcription, asymmetry, fraction of T4 late RNA, and sensitivity to inhibition by rifampicin and streptolydigin) are described.