In vitro analysis of transcriptional activators and polyadenylation
Takashi Nagaike1, James L Manley
1Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba-shi, Ibaraki, Japan.
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 identification 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.
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