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. 2021 May 18;120(10):1883-1893.
doi: 10.1016/j.bpj.2021.03.007. Epub 2021 Mar 16.

The N-terminal domain of the A12.2 subunit stimulates RNA polymerase I transcription elongation

Catherine E Scull  1 , Aaron L Lucius  2 , David A Schneider  3
Affiliations

The N-terminal domain of the A12.2 subunit stimulates RNA polymerase I transcription elongation

Catherine E Scull et al. Biophys J. .

Abstract

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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Figures

Figure 1
Figure 1
RNA polymerase I subunit A12 contains an intrinsic cleavage factor. (A) Sequence conservation among the eukaryotic Pols adapted from (16) (sequences not to scale). (B) The A12 subunit of Pol I (shown in the surface filled model) is composed of a paralog of Pol II’s Rpb9 subunit (green) and a paralog of Pol II transcription factor TFIIS (yellow). The image was made in PyMOL using the Protein Data Bank structure, PDB: 4C2M (17). To see this figure in color, go online.
Figure 2
Figure 2
ΔA12CTD Pol I lacks the ability to cleave nascent RNA. (A) Cartoon schematic of single-nucleotide addition experimental strategy displays mixing of radiolabeled 10-mer elongation complex with ATP and Mg2+ over time to produce 11-mer and CA dinucleotide radiolabeled RNA species. (B) Time courses were carried out from 5 ms to 10 s for WT Pol I, and extension of 10-mer RNA to 11-mer RNA is observed. Subsequent cleavage of the 11-mer species can be measured by the appearance of the CA dinucleotide species. The GC species is produced during labeling of the RNA and thus is constant throughout the time course. Time courses were likewise carried out from 5 ms to 10 s for (C) ΔA12 and (D) ΔA12CTD Pol I, and extension of 10-mer RNA to 11-mer RNA was observed. To see this figure in color, go online.
Figure 3
Figure 3
ΔA12CTD Pol I single nucleotide addition is faster than WT and ΔA12 Pol I: (A) time courses were quantified for WT Pol I (black), ΔA12 Pol I (gray, data from (18)), and ΔA12CTD Pol I (green). WT, ΔA12, and ΔA12CTD Pol I time courses were fitted to the sum of two exponential curves. Rate constants (kobs1 and kobs2) were extracted from fits; error represents deviation about the mean for three independent replicates. Rate constants were calculated for each [ATP] for (B) kobs1 for WT Pol I (the rise phase), (C and D) kobs1 and kobs2 for ΔA12 Pol I (18), and (E and F) kobs1 and kobs2 for ΔA12CTD Pol I. (B–E) Data were fitted to a rectangular hyperbola (Eq. 5), and parameters kmax and K1/2 were calculated (R2 = 0.998). The x axes were plotted in log space for visualization. Error was evaluated at a 95% confidence interval. (F) kobs2 values for ΔA12CTD Pol I were plotted over a series of [ATP]s; kobs2ΔA12CTD Pol I data did not show a dependence on [ATP] (R2 = 0.49). To see this figure in color, go online.
Figure 4
Figure 4
Global fitting of ΔA12CTD Pol I single-nucleotide addition time courses to scheme 2. ΔA12CTD Pol I time courses were fitted to scheme 2 such that elementary rate constants could be derived to describe single-nucleotide addition. Error bars represent error about the mean for three independent replicates. Derived rate constants are enumerated in Table 2. To see this figure in color, go online.
Figure 5
Figure 5
The ΔA12CTD mutation does not result in altered stability of Pol I elongation complexes. (A) Radiolabeled elongation complexes are mixed with destabilizing [KCl] and RNase A and are then allowed to incubate for a given time = t. Elongation complexes that are not disrupted by [KCl] will result in a protected 10-mer species, whereas elongation complexes that are disrupted by [KCl] will fall apart, releasing RNA for degradation by RNase A. (B) 28% denaturing urea PAGE is used to separate 10-mer and 7-mer species for quantification. (C) Normalized fraction of RNA degraded for WT Pol I (black) and ΔA12CTD Pol I (green) is plotted over time. Error bars represent standard deviation about the mean for 3 replicates. No significant difference in elongation complex stability was noted between WT and ΔA12CTD Pol I. To see this figure in color, go online.
Figure 6
Figure 6
ΔA12CTD Pol I is sensitive to downstream DNA sequence. (A) Abridged schematic displaying the altered downstream template sequence used for experiments (green, high downstream GC template, full sequence displayed in Fig. S1) was altered to include a high-AT downstream region (yellow, high downstream AT template). (B) Single-nucleotide experiments were done at saturating ATP (1 mM), and quantification of transcription elongation between the high downstream GC template (black) and high downstream AT template (yellow) displays that ΔA12CTD Pol I is sensitive to downstream DNA sequence. Data were fitted to the sum of two exponential curves (Eq. 2; Table S2), and error bars represent standard deviation about the mean for three replicates. To see this figure in color, go online.
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