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J Vis Exp. 2019 May 13;(147). doi: 10.3791/59497.

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events.

Author information

1
Stowers Institute for Medical Research.
2
Stowers Institute for Medical Research; Department of Biochemistry and Molecular Biology, Kansas University Medical Center.
3
Stowers Institute for Medical Research; Department of Biochemistry and Molecular Biology, Kansas University Medical Center; RCC@stowers.org.

Abstract

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3'-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility. In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

PMID:
31132066
DOI:
10.3791/59497

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