Analysis of Activator-Dependent Transcription Reinitiation In Vitro

Abstract

This chapter aims to describe the technical details of a set of methods collectively capable of distinguishing between transcription initiation and reinitiation and measuring the initiation rate in cell-free transcription systems. These procedures were developed to study heat shock gene regulation. The chapter focuses to provide a detailed description of the methodology. The study relied on the availability of a powerful cell-free transcription system, capable of supporting multiple initiation events. Prior to induction, heat shock promoters are characterized by a paused polymerase: an RNA polymerase II complex has been recruited to the promoter, has initiated transcription, but is arrested to pause elongation some 25 nucleotides downstream of the transcription start site. Heat shock conditions induce the interaction of HSF with the promoter and a concomitant release of the polymerase to enter the elongation phase. Pausing of the polymerase is controlled by upstream activators, and the release of the first polymerase is followed by very rapid initiation cycles. Results suggested that these subsequent initiation events are indeed reinitiation cycles.