Protocol to detect spontaneous termination of yeast RNAPII transcription in vitro

Abstract

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• molecular biology
• gene expression
• protein biochemistry