Scull Catherine E, Lucius Aaron L, Schneider David A
The Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama.
the Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama.
Biophys J. 2021 May 18;120(10):1883-1893. doi: 10.1016/j.bpj.2021.03.007. Epub 2021 Mar 16.
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.
真核生物表达三种依赖DNA的RNA聚合酶（Pol），它们负责细胞基因组表达的全部过程。这三种Pol已经进化到能够表达特定的RNA群体，因此在结构和功能上都有所分化，以有效地执行其特定的转录作用。这种分化的一个例子是，Pol I包含一个校对因子作为真正的亚基，而Pol II则招募转录因子TFIIS进行校对。Pol I的A12.2（A12）亚基与Pol II的Rpb9亚基以及转录因子TFIIS具有同源性，TFIIS可促进Pol II的RNA切割和校对。在本研究中，通过突变分析探究了A12亚基的TFIIS样C末端结构域和Rpb9样N末端结构域的功能贡献。我们发现，缺乏A12亚基C末端结构域的Pol I突变体（ΔA12CTD Pol I）在单核苷酸添加方面比野生型Pol I略快，但ΔA12CTD Pol I缺乏RNA切割活性。ΔA12CTD Pol I在延伸复合物稳定性方面也与野生型Pol I相似，而之前已证明去除整个A12亚基（ΔA12 Pol I）可稳定转录延伸复合物。此外，ΔA12CTD Pol I对下游序列背景敏感，因为暴露于富含AT的下游DNA的ΔA12CTD Pol I比ΔA12 Pol I更容易发生停滞。这些数据表明，A12的N末端结构域不会刺激Pol I的内在RNA切割活性，而是有助于Pol I的核心转录延伸特性。
