Research Article 13: Cell-Free Studies on the Regulation of the lac Operon

Abstract

INTRODUCTION
The ideal test for a model of gene regulation would be the measurement of gene activity as a function of the concentration of each of the alleged regulating components. A cell-free system in which it would be possible to mix the required components in any amounts and observe the effects on gene activity would be most suitable for such studies. About four years ago our laboratory began developing such a cell-free system for the lac operon. At a minimum, demonstration of gene activity would require a system containing DNA, RNA polymerase, the substrates and cofactors for RNA synthesis, and a procedure for characterization of the RNA. In vitro systems that synthesize only the immediate RNA product of the gene have thus far proven ineffective for bacterial gene studies; for some unknown reason RNA polymerase in these systems does not function with sufficient discrimination. The result is a chaotic assemblage of RNA molecules whose synthesis is insensitive to in vivo gene regulating factors (Zubay, unpublished results).

Thus, the immediate challenge in developing a workable cell-free system lies in finding conditions under which RNA polymerase recognizes only true starts and true stops on the gene. In the cell-free system which we have developed, DNA-directed synthesis of the proteins of the lac operon has been studied. This system is quite complex, comprising a cell-free extract of E. coli, DNA from the defective transducing virus ϕ80dlac (although any DNA containing the lac operon is active) and the cofactors and substrates necessary for...