Reversible Kinetics in Multi-nucleotide Addition Catalyzed by S. cerevisiae RNA polymerase II Reveal Slow Pyrophosphate Release

Kaila B Fuller; Ruth Q Jacobs; David A Schneider; Aaron L Lucius

doi:10.1016/j.jmb.2024.168606

Reversible Kinetics in Multi-nucleotide Addition Catalyzed by S. cerevisiae RNA polymerase II Reveal Slow Pyrophosphate Release

J Mol Biol. 2024 May 8;436(12):168606. doi: 10.1016/j.jmb.2024.168606. Online ahead of print.

Authors

Kaila B Fuller¹, Ruth Q Jacobs², David A Schneider³, Aaron L Lucius⁴

Affiliations

¹ Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
² Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
³ Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address: dschneid@uab.edu.
⁴ Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address: allucius@uab.edu.

PMID: 38729258
DOI: 10.1016/j.jmb.2024.168606

Abstract

Eukaryotes express at least three nuclear DNA dependent RNA polymerases (Pols). Pols I, II, and III synthesize ribosomal (r) RNA, messenger (m) RNA, and transfer (t) RNA, respectively. Pol I and Pol III have intrinsic nuclease activity conferred by the A12.2 and C11 subunits, respectively. In contrast, Pol II requires the transcription factor (TF) IIS to confer robust nuclease activity. We recently reported that in the absence of the A12.2 subunit Pol I reverses bond formation by pyrophosphorolysis in the absence of added PPi, indicating slow PPi release. Thus, we hypothesized that Pol II, naturally lacking TFIIS, would reverse bond formation through pyrophosphorolysis. Here we report the results of transient-state kinetic experiments to examine the addition of nine nucleotides to a growing RNA chain catalyzed by Pol II. Our results indicate that Pol II reverses bond formation by pyrophosphorolysis in the absence of added PPi. We propose that, in the absence of endonuclease activity, this bond reversal may represent kinetic proofreading. Thus, given the hypothesis that Pol I evolved from Pol II through the incorporation of general transcription factors, pyrophosphorolysis may represent a more ancient form of proofreading that has been evolutionarily replaced with nuclease activity.

Keywords: RNA polymerase II; pyrophosphoralysis; transient-state kinetics.