Search

Eukaryotes express three DNA-dependent RNA polymerases (Pols) that are responsible for the entirety of cellular genomic expression. The three Pols have evolved to express specific cohorts of RNAs and thus have diverged both structurally and functionally to efficiently execute their specific transcriptional roles. One example of this divergence is Pol I’s inclusion of a proofreading factor as a bona fide subunit, as opposed to Pol II, which recruits a transcription factor, TFIIS, for proofreading. The A12.2 (A12) subunit of Pol I shares homology with both the Rpb9 subunit of Pol II as well as the transcription factor TFIIS, which promotes RNA cleavage and proofreading by Pol II. In this study, the functional contribution of the TFIIS-like C-terminal domain and the Rpb9-like N-terminal domain of the A12 subunit are probed through mutational analysis. We found that a Pol I mutant lacking the C-terminal domain of the A12 subunit (ΔA12CTD Pol I) is slightly faster than wild-type Pol I in single-nucleotide addition, but ΔA12CTD Pol I lacks RNA cleavage activity. ΔA12CTD Pol I is likewise similar to wild-type Pol I in elongation complex stability, whereas removal of the entire A12 subunit (ΔA12 Pol I) was previously demonstrated to stabilize transcription elongation complexes. Furthermore, the ΔA12CTD Pol I is sensitive to downstream sequence context, as ΔA12CTD Pol I exposed to AT-rich downstream DNA is more arrest prone than ΔA12 Pol I. These data demonstrate that the N-terminal domain of A12 does not stimulate Pol I intrinsic RNA cleavage activity, but rather contributes to core transcription elongation properties of Pol I.

Despite sharing a highly conserved core architecture with their prokaryotic counterparts, eukaryotic multisubunit RNA polymerases (Pols) have undergone structural divergence and biological specialization. Interesting examples of structural divergence are the A12.2 and C11 subunits of Pols I and III, respectively. Whereas all known cellular Pols possess cognate protein factors that stimulate cleavage of the nascent RNA, Pols I and III have incorporated their cleavage factors as bona fide subunits. Although it is not yet clear why these polymerases have incorporated their cleavage factors as subunits, a picture is emerging that identifies roles for these subunits beyond providing nucleolytic activity. Specifically, it appears that both A12.2 and C11 are required for efficient termination of transcription by Pols I and III. Given that termination involves destabilization of the elongation complex (EC), we tested whether A12.2 influences stability of the Pol I EC. Using, to our knowledge, a novel assay to measure EC dissociation kinetics, we have determined that A12.2 is an intrinsic destabilizer of the Pol I EC. In addition, the salt concentration dependence of Pol I EC dissociation kinetics suggests that A12.2 alters electrostatic interactions within the EC. Importantly, these data present a mechanistic basis for the requirement of A12.2 in Pol I termination. Combined with recent work demonstrating the direct involvement of A12.2 in Pol I nucleotide incorporation, this study further supports the concept that A12.2 cannot be viewed solely as a cleavage factor.