RNA polymerase I (Pol I) lobe-binding subunit Rpa12.2 promotes RNA cleavage and proofreading.

Elongating nuclear RNA polymerases (Pols) frequently pause, backtrack, and are then reactivated by endonucleolytic cleavage. Pol backtracking and RNA cleavage are also crucial for proofreading, which contributes to transcription fidelity. RNA polymerase I (Pol I) of the yeast Saccharomyces cerevisiae synthesizes exclusively 35S rRNA, the precursor transcript of mature ribosomal 5.8S, 18S, and 25S rRNA. Pol I contains the specific heterodimeric subunits Rpa34.5/49 and subunit Rpa12.2, which have been implicated in RNA cleavage and elongation activity, respectively. These subunits are associated with the Pol I lobe structure and encompass different structural domains, but the contribution of these domains to RNA elongation is unclear. Here, we used Pol I mutants or reconstituted Pol I enzymes to study the effects of these subunits and/or their distinct domains on RNA cleavage, backtracking, and transcription fidelity in defined in vitro systems. Our findings suggest that the presence of the intact C-terminal domain of Rpa12.2 is sufficient to support the cleavage reaction, but that the N-terminal domains of Rpa12.2 and the heterodimer facilitate backtracking and RNA cleavage. Since both N-terminal and C-terminal domains of Rpa12.2 were also required to faithfully incorporate NTPs in the growing RNA chain, efficient backtracking and RNA cleavage might be a prerequisite for transcription fidelity. We propose that RNA Pols containing efficient RNA cleavage activity are able to add and remove nucleotides until the matching nucleotide supports RNA chain elongation, whereas cleavage-deficient enzymes can escape this proofreading process by incorporating incorrect nucleotides.

Features of yeast RNA polymerase I with special consideration of the lobe binding subunits.

Ribosomal RNAs (rRNAs) are structural components of ribosomes and represent the most abundant cellular RNA fraction. In the yeast Saccharomyces cerevisiae, they account for more than 60 % of the RNA content in a growing cell. The major amount of rRNA is synthesized by RNA polymerase I (Pol I). This enzyme transcribes exclusively the rRNA gene which is tandemly repeated in about 150 copies on chromosome XII. The high number of transcribed rRNA genes, the efficient recruitment of the transcription machinery and the dense packaging of elongating Pol I molecules on the gene ensure that enough rRNA is generated. Specific features of Pol I and of associated factors confer promoter selectivity and both elongation and termination competence. Many excellent reviews exist about the state of research about function and regulation of Pol I and how Pol I initiation complexes are assembled. In this report we focus on the Pol I specific lobe binding subunits which support efficient, error-free, and correctly terminated rRNA synthesis.

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

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.