Single-Molecule Biochemistry Lab, Institute of Microbiology and Archaea Centre, University of Regensburg, 93053, Regensburg, Germany.
Department of Chemistry and Center for NanoScience (CeNS), Ludwig-Maximilians-Universität München, 80539, München, Germany.
Nat Commun. 2020 Jun 5;11(1):2828. doi: 10.1038/s41467-020-16702-x.
The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.
TATA 结合蛋白（TBP）和转录因子（TF）IIB 样因子是所有真核起始复合物的重要组成部分。然而，RNA 聚合酶（RNAP）III 系统中额外起始因子 Bdp1 的出现和严格要求的原因仍然难以捉摸。在这方面，一个研究较少的方面是 DNA 压缩和转录活性引起的 DNA 应变对起始复合物形成的影响。我们利用基于 DNA 折纸的力夹在皮牛顿力下在单细胞水平上跟踪 RNAP II 和 RNAP III 系统中人类起始复合物的组装。我们证明 TBP-DNA 复合物对力敏感，TFIIB 足以稳定 TBP 在应变启动子上的结合。相比之下，Bdp1 是确保起始因子（因此也是聚合酶本身）在 RNAP III 系统中稳定锚定的关键组成部分。因此，我们为 Bdp1 对 RNAP III 高转录输出的关键作用提供了一个解释。
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