在果蝇的 U6 snRNA 基因启动子上组装 SNAPc、Bdp1 和 TBP。
Assembly of SNAPc, Bdp1, and TBP on the U6 snRNA Gene Promoter in Drosophila melanogaster.
机构信息
Department of Chemistry and Biochemistry and Molecular Biology Institute, San Diego State University, San Diego, California, USA.
Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, California, USA.
出版信息
Mol Cell Biol. 2020 May 28;40(12). doi: 10.1128/MCB.00641-19.
U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previously, we found that SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. Here, we show that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and we identify a region of Bdp1 that is sufficient for TBP recruitment. Moreover, we find that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed us to build a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP.
U6 snRNA 由 RNA 聚合酶 III（Pol III）转录，具有外部上游启动子，该启动子由 Pol III 基本转录因子 IIIB 的 TBP 亚基识别的 TATA 序列和由小核 RNA 激活蛋白复合物（SNAPc）识别的近端序列元件（PSE）组成。此前，我们发现 SNAPc（DmSNAPc）与 U6 PSE 的结合可以募集 Pol III 一般转录因子 Bdp1 与 DNA 形成稳定的复合物。在这里，我们表明 DmSNAPc-Bdp1 可以招募 TBP 到 U6 启动子，并且我们确定了 Bdp1 的一个区域，该区域足以进行 TBP 募集。此外，我们发现 Bdp1 的同一区域与 U6 PSE 内的核苷酸交联，这些位置也与 DmSNAPc 交联。最后，交联质谱揭示了特定的 DmSNAPc 亚基与 Bdp1 和 TBP 的可能相互作用。这些数据以及先前的发现使我们能够构建一个更全面的 DmSNAPc-Bdp1-TBP 复合物在 U6 启动子上的模型，该模型包括几乎所有的 DmSNAPc、Bdp1 的一部分和 TBP 的保守区。
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