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

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.

Cytosine base modifications regulate DNA duplex stability and metabolism

“…The majority of the RNA produced in cells is produced by RNA polymerase I and, hence, this enzyme better represents the measurements in vivo (see below). To allow nonspecific RNA polymerase initiation, we generated templates containing a 3′-overhang (3′-tailed templates) based on the nicking enzyme Nb.BsmI ( 46 ). Slot blot analyses with antibodies specifically recognizing 5mC and 5hmC, respectively, confirmed the modification of the templates ( Supplementary Figure S13A ).…”

“…Saccharomyces cerevisiae DNA-dependent RNA polymerase (Pol) I was purified as described ( 44 , 45 ). The polymerization assay is based on previously published protocols ( 46 ). Per transcription reaction 0.25 pmol Pol I, 0.125 pmol template (described above), 50 pmol GpC dinucleotide and HEPES (pH 7.8; end concentration 50 mM) were mixed.…”

Conserved strategies of RNA polymerase I hibernation and activation

“…The subcomplex shows similarities to initiation factors TFIIF and TFIIE in the Pol II transcription system, as suggested from homology to crystal structures and native mass spectrometry analysis 30 . However, the A49/A34.5 subcomplex has also been described as important for Pol I elongation 20 , 31 , 32 and is constantly attached to the Pol I core throughout elongation in vivo 33 . Dissociation of the subcomplex from Sc Pol I was observed under specific experimental conditions in vitro 20 or in ECs established using a nucleotide analogon 21 .…”

Coupling Between Production of Ribosomal RNA and Maturation: Just at the Beginning

“…In the absence of Rpa12, Pol I catalytic properties are affected ( Appling et al, 2018 ; Scull et al, 2021 ). Furthermore, Pol I transcription through a linear mono-nucleosomal template was shown to be defective in the absence of the lobe-binding subunits ( Merkl et al, 2020 ). Mutations affecting the Rpa135 subunit were also shown to affect transcription elongation.…”