Abortive Initiation of In Vitro RNA Synthesis on Bacteriophage λ DNA

Abstract

INTRODUCTION
The initiation of RNA synthesis is an important control point in gene expression. Both negative and positive controls of the initiation process have evolved that allow bacteria and bacteriophage to adapt rapidly to changing environments or to program a complex temporal pattern of RNA and thus protein synthesis. The study of in vitro initiation and its control has been complicated by the unique character of the process itself; that is, initiation occurs only once for each high molecular weight RNA chain synthesized. RNA polymerase clearly spends most of its time elongating RNA. Therefore in order to study the initiation phase of RNA synthesis directly, we require either a high-speed sampling technique or some other method that will amplify the biochemical events that result in initiation. In the early stages of a study that had as its goal the development of the high-speed sampling assay, we discovered that it was possible to amplify certain steps in the initiation process. We define initiation as the substrate binding and catalytic steps leading to the formation of the first phosphodiester bond; the method we describe in this paper allows a convenient steady-state approach to be employed in the study of RNA initiation.

Most of the experiments described in this paper have been done with λb2 DNA and E. coli RNA polymerase. We chose the λ template for several reasons. At the biochemical level it is perhaps the best characterized genome known. The four major in vitro RNA products, represented schematically in Figure 1...