Kang Yoon Soon, Kurano Michelle, Stumph William E
Department of Chemistry and Biochemistry, Molecular Biology Institute, San Diego State University, San Diego, CA 92182-1030, USA.
Department of Biology, Molecular Biology Institute, San Diego State University, San Diego, CA 92182-1030, USA.
Nucleic Acids Res. 2014 Nov 10;42(20):12440-54. doi: 10.1093/nar/gku905. Epub 2014 Oct 16.
The small nuclear RNA (snRNA) activating protein complex (SNAPc) is essential for transcription of genes that encode the snRNAs. Drosophila melanogaster SNAPc (DmSNAPc) consists of three subunits (DmSNAP190, DmSNAP50 and DmSNAP43) that form a stable complex that recognizes an snRNA gene promoter element called the PSEA. Although all three subunits are required for sequence-specific DNA binding activity, only DmSNAP190 possesses a canonical DNA binding domain consisting of 4.5 tandem Myb repeats homologous to the Myb repeats in the DNA binding domain of the Myb oncoprotein. In this study, we use site-specific protein-DNA photo-cross-linking technology followed by site-specific protein cleavage to map domains of DmSNAP190 that interact with specific phosphate positions in the U6 PSEA. The results indicate that at least two DmSNAP190 Myb repeats contact the DNA in a significantly different manner when DmSNAPc binds to a U6 PSEA versus a U1 PSEA, even though the two PSEA sequences differ at only 5 of 21 nucleotide positions. The results are consistent with a model in which the specific DNA sequences of the U1 and U6 PSEAs differentially alter the conformation of DmSNAPc, leading to the subsequent recruitment of different RNA polymerases to the U1 and U6 gene promoters.
小核RNA(snRNA)激活蛋白复合体(SNAPc)对于编码snRNA的基因转录至关重要。果蝇SNAPc(DmSNAPc)由三个亚基(DmSNAP190、DmSNAP50和DmSNAP43)组成,它们形成一个稳定的复合体,识别一种名为PSEA的snRNA基因启动子元件。尽管所有三个亚基对于序列特异性DNA结合活性都是必需的,但只有DmSNAP190拥有一个由4.5个串联Myb重复序列组成的典型DNA结合结构域,该结构域与Myb癌蛋白DNA结合结构域中的Myb重复序列同源。在本研究中,我们使用位点特异性蛋白质-DNA光交联技术,随后进行位点特异性蛋白质切割,以绘制DmSNAP190与U6 PSEA中特定磷酸位置相互作用的结构域。结果表明,当DmSNAPc与U6 PSEA结合时,至少两个DmSNAP190 Myb重复序列与DNA的接触方式与与U1 PSEA结合时显著不同,尽管这两个PSEA序列在21个核苷酸位置中只有5个不同。这些结果与一个模型一致,即U1和U6 PSEAs的特定DNA序列差异地改变DmSNAPc的构象,导致随后不同的RNA聚合酶被招募到U1和U6基因启动子上。