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.

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Journal Article | Research Support, N.I.H., Extramural | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Scull CE, Lucius AL, Schneider DA

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