The A12.2 Subunit Plays an Integral Role in Pyrophosphate Release of RNA Polymerase I.

Autor: Fuller KB; Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA., Jacobs RQ; Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA., Schneider DA; Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address: dschneid@uab.edu., Lucius AL; Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address: allucius@uab.edu.
Jazyk: angličtina
Zdroj: Journal of molecular biology [J Mol Biol] 2023 Aug 01; Vol. 435 (15), pp. 168186. Date of Electronic Publication: 2023 Jun 22.
DOI: 10.1016/j.jmb.2023.168186
Abstrakt: RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA), which is the first and rate-limiting step in ribosome biosynthesis. A12.2 (A12) is a critical subunit of Pol I that is responsible for activating Pol I's exonuclease activity. We previously reported a kinetic mechanism for single-nucleotide incorporation catalyzed by Pol I lacking the A12 subunit (ΔA12 Pol I) purified from S. cerevisae and revealed that ΔA12 Pol I exhibited much slower incorporation compared to Pol I. However, it is unknown if A12 influences each nucleotide incorporation in the context of transcription elongation. Here, we show that A12 contributes to every repeating cycle of nucleotide addition and that deletion of A12 results in an entirely different kinetic mechanism compared to WT Pol I. We found that instead of one irreversible step between each nucleotide addition cycle, as reported for wild type (WT) Pol I, the ΔA12 variant requires one reversible step to describe each nucleotide addition. Reversibility fundamentally requires slow PPi release. Consistently, we show that Pol I is more pyrophosphate (PPi) concentration dependent than ΔA12 Pol I. This observation supports the model that PPi is retained in the active site of ΔA12 Pol I longer than WT Pol I. These results suggest that A12 promotes PPi release, revealing a larger role for the A12.2 subunit in the nucleotide addition cycle beyond merely activating exonuclease activity.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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