Protocol for monitoring and analyzing single nucleotide incorporation by S. cerevisiae RNA polymerases

Ruth Q Jacobs; Nathan F Bellis; Aaron L Lucius; David A Schneider

doi:10.1016/j.xpro.2023.102191

Protocol for monitoring and analyzing single nucleotide incorporation by S. cerevisiae RNA polymerases

STAR Protoc. 2023 Mar 24;4(2):102191. doi: 10.1016/j.xpro.2023.102191. Online ahead of print.

Authors

Ruth Q Jacobs¹, Nathan F Bellis¹, Aaron L Lucius², David A Schneider³

Affiliations

¹ Deparment of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
² Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address: allucius@uab.edu.
³ Deparment of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address: dschneid@uab.edu.

Abstract

Here we present an optimized protocol for monitoring and analyzing single nucleotide incorporation by RNA polymerases. This protocol describes the assembly of Saccharomyces cerevisiae RNA polymerase I elongation complexes in a promoter-independent system in vitro. We describe how to collect a time course using a quench-flow, a rapid mixing instrument, and subsequently resolve reactions on a polyacrylamide gel. Finally, we detail how to quantify the gel images. For complete details on the use and execution of this protocol, please refer to Appling et al. (2015).¹.

Keywords: Cell Biology; Chemistry; Genetics; Model Organisms; Molecular Biology; Protein Biochemistry; Protein Expression and Purification.

Abstract

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