Assays for Transcription Factor Activity

Most transcription activator proteins have three important features that can be probed at the molecular level: They bind to specific sequences near promoters, they can be interconverted between active and inactive forms by covalent or noncovalent modification, and, when bound at target promoters, they can stimulate the initiation of transcription by RNA polymerase (1 ). This chapter is concerned with in vitro methods for measuring the transcription activation function of this important class of proteins. In most cases these methods will be applied to activator proteins that have been substantially purified, and in which the target promoter sequence is known and the binding site characterized. Chapters l–l1 in this volume cover methods that can be exploited to locate and investigate specific binding sites for activators. Here we are concerned with the measurement of the products of activation. Since Escherichia coli transcription activators have been studied more than any other, we will take E. coli systems as the paradigm. In principle the techniques can be applied to any organism from which in vitro systems have been developed.