In vitro system for middle T4 RNA. I. Studies with Escherichia coli RNA polymerase.

We describe crude in vitro systems from T4-infected cells which reflect in vivo T4 regulation. Lysates from cells which had been infected in the presence of chloramphenicol manifest the same polarity of RNA synthesis as did the infected cells. Next, we describe a complementation system between lysates which have no RNA synthetic capacity and purified RNA polymerase; in this system, delayed early RNA synthesis in vitro depends on the presence of an active mot gene product. Mot activity controls middle mode gene expression in vivo. In vitro, not activity in the lysate directs RNA polymerase to initiate on regions of DNA that are otherwise inaccessible. This mot-dependent delayed early RNA synthesis in vitro is seen at 0.1 and 0.2 M KCl, but not at 0.05 M KCl. We present a model in which mot is a DNA melting protein necessary for recognition of a middle promoters by either Escherichia coli or T4-modified RNA polymerase which contains E. coli sigma subunit.