Protocol to detect spontaneous termination of yeast RNAPII transcription in vitro

Zhong Han; Jesper Q Svejstrup

doi:10.1016/j.xpro.2024.103369

Protocol to detect spontaneous termination of yeast RNAPII transcription in vitro

STAR Protoc. 2024 Dec 20;5(4):103369. doi: 10.1016/j.xpro.2024.103369. Epub 2024 Oct 10.

Authors

Zhong Han¹, Jesper Q Svejstrup²

Affiliations

¹ Department of Cellular and Molecular Medicine, Panum Institute, Blegdamsvej 3B, University of Copenhagen, 2200 Copenhagen N, Denmark. Electronic address: zhonghan@sund.ku.dk.
² Department of Cellular and Molecular Medicine, Panum Institute, Blegdamsvej 3B, University of Copenhagen, 2200 Copenhagen N, Denmark. Electronic address: jsvejstrup@sund.ku.dk.

PMID: 39392748
DOI: 10.1016/j.xpro.2024.103369

Abstract

We present a protocol for the in vitro detection of spontaneous termination of yeast RNA polymerase II (RNAPII) transcription using bead-immobilized elongation complexes (ECs). We describe the steps for EC assembly, ligation to a long transcription template, and the in vitro elongation and termination reactions. Our protocol has proven successful for identifying spontaneous termination in the yeast CYC1 terminator. For complete details on the use and execution of this protocol, please refer to Han et al.¹.

Keywords: gene expression; molecular biology; protein biochemistry.

MeSH terms

RNA Polymerase II* / genetics
RNA Polymerase II* / metabolism
Saccharomyces cerevisiae Proteins / genetics
Saccharomyces cerevisiae Proteins / metabolism
Saccharomyces cerevisiae* / genetics
Transcription Termination, Genetic
Transcription, Genetic / genetics

Substances

RNA Polymerase II
Saccharomyces cerevisiae Proteins