RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure

J Biol Chem. 2020 Apr 10;295(15):4782-4795. doi: 10.1074/jbc.RA119.011827. Epub 2020 Feb 14.

Abstract

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I-associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I-specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.

Keywords: RNA polymerase I; RNA polymerase II; RNA polymerase III; Rpa34.5; Rpa49; chromatin; nucleolus; nucleosome; ribosomal ribonucleic acid (rRNA) (ribosomal RNA); ribosome; transcription; transcription elongation factor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatin / genetics
  • Chromatin / metabolism*
  • DNA Replication
  • DNA, Ribosomal / genetics
  • DNA, Ribosomal / metabolism
  • Nucleosomes / genetics
  • Nucleosomes / metabolism*
  • Promoter Regions, Genetic
  • Protein Binding
  • Protein Subunits / metabolism
  • RNA Polymerase I / chemistry
  • RNA Polymerase I / genetics
  • RNA Polymerase I / metabolism*
  • RNA Polymerase II / chemistry
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • RNA Polymerase III / chemistry
  • RNA Polymerase III / genetics
  • RNA Polymerase III / metabolism*
  • Ribosomes
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism*
  • Transcription, Genetic*

Substances

  • Chromatin
  • DNA, Ribosomal
  • Nucleosomes
  • Protein Subunits
  • RNA Polymerase II
  • RNA Polymerase I
  • RNA Polymerase III

Associated data

  • PDB/5M3F
  • PDB/4C2M
  • PDB/5F8J
  • PDB/8W6S
  • PDB/1Y1W