The N-terminal domain of the A12.2 subunit stimulates RNA polymerase I transcription elongation
The N-terminal domain of the A12.2 subunit stimulates RNA polymerase I transcription elongation
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DOI：
10.1016/j.bpj.2021.03.007
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发表时间：
2021-05-18
影响因子：
3.4
通讯作者：
Schneider, David A.
中科院分区：
文献类型：
--
作者：
Scull, Catherine E.;Lucius, Aaron L.;Schneider, David A.
真核生物表达三种DNA依赖性RNA聚合酶（Pos），负责整个细胞基因组表达。这三种Pol已经进化为表达特定的RNA组群，因此在结构和功能上都有分歧，以有效地执行其特定的转录作用。这种分歧的一个例子是Pol I包含了一个校对因子作为真正的亚基，而Pol II则招募了一个转录因子TFIIS来校对。Pol I的A12.2（A12）亚基与Pol II的Rpb 9亚基以及转录因子TFIIS共享同源性，TFIIS促进RNA切割和Pol II的校对。在这项研究中，TFIIS样的C-末端结构域和Rpb 9样的N-末端结构域的A12亚基的功能的贡献进行了探测，通过突变分析。我们发现缺乏A12亚基C-末端结构域的Pol I突变体（Delta 12 CTD Pol I）在单核苷酸添加中比野生型Pol I稍快，但Delta A12 CTD Pol I缺乏RNA切割活性。Δ A12 CTD Pol I在延伸复合物稳定性方面同样类似于野生型Pol I，而先前证明去除整个A12亚基（Δ A12 Pol I）可稳定转录延伸复合物。此外，Delta A12 CTD Pol I对下游序列环境敏感，因为暴露于富含AT的下游DNA的Delta A12 CTD Pol I比Delta A12 Pol I更容易停滞。这些数据表明，A12的N-末端结构域不刺激Pol I固有的RNA切割活性，而是有助于Pol I的核心转录延伸性质。
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 (Delta 12CTD Pol I) is slightly faster than wild-type Pol I in single-nucleotide addition, but Delta A12CTD Pol I lacks RNA cleavage activity. Delta A12CTD Pol I is likewise similar to wild-type Pol I in elongation complex stability, whereas removal of the entire A12 subunit (Delta A12 Pol I) was previously demonstrated to stabilize transcription elongation complexes. Furthermore, the Delta A12CTD Pol I is sensitive to downstream sequence context, as Delta A12CTD Pol I exposed to AT-rich downstream DNA is more arrest prone than Delta A12 Pol I. These data demonstrate that the N-terminal domain of Al2 does not stimulate Pol I intrinsic RNA cleavage activity, but rather contributes to core transcription elongation properties of Pol I.