In vitro analysis of transcriptional activators and polyadenylation

Abstract

In vitro assays have provided a valuable tool to study the mechanism of 3′ processing of eukaryotic mRNA precursors and have contributed a great deal to the identifi cation of factors that carry out and regulate 3′ processing. Previously, we have shown that transcriptional activators directly enhance polyadenylation by utilizing in vitro transcription-coupled polyadenylation with the prototypical transcription activator GAL4-VP16. In this chapter, we describe a detailed protocol for this assay, which will be useful in examining potential roles for other transcription-related factors in 3′ processing and other questions related to the coupling of transcription and mRNA polyadenylation.