Characterization of the mechanisms of transcription termination by the helicase Sen1

Abstract

In vitro transcription systems have been widely used to study all the steps of transcription from initiation to termination and many transcription-coupled processes. Here we describe an in vitro transcription-termination assay that we have used for the analysis of the mechanism of termination by the yeast helicase Sen1. In this system, we use highly purifi ed proteins to assemble ternary elongation complexes (RNA polymerase, DNA template, and nascent RNA) on biotinylated DNA that is subsequently immobilized on streptavidin beads. After allowing transcription by the addition of nucleotides, the termination events can be detected and quantifi ed by comparing the amounts of polymerases and transcripts released from the DNA templates in reactions performed in the absence or in the presence of purifi ed Sen1. By modifying different parameters of the assay, this technique allows the study of several aspects of the termination reaction.