Architectural arrangement of cloned proximal sequence element-binding protein subunits on Drosophila U1 and U6 snRNA gene promoters
Architectural arrangement of cloned proximal sequence element-binding protein subunits on Drosophila U1 and U6 snRNA gene promoters
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DOI：
10.1128/mcb.24.5.1897-1906.2004
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发表时间：
2004-03-01
影响因子：
5.3
通讯作者：
Stumph, WE
中科院分区：
文献类型：
--
作者：
Li, C;Harding, GA;Stumph, WE
RNA 聚合酶 11（U1 至 U5）或 RNA 聚合酶 III（U6）对 snRNA 基因的转录取决于位于转录起始位点上游约 40 至 60 bp 的近端序列元件 (PSE)。在果蝇中，RNA 聚合酶的特异性由保守的 21 bp PSE 中少至三个核苷酸的差异决定。先前的光交联研究表明，黑腹果蝇 PSE 结合蛋白 DmPBP 包含三个亚基（DmPBP45、DmPBP49 和 DmPBP95），这些亚基与 DNA 结合形成构象不同的复合物，具体取决于该蛋白是与 U1 还是 U6 PSE 结合。我们已经鉴定并克隆了编码 DmPBP 这些亚基的基因，因为它们与 SNAP 的五个亚基（人类 PBP）中的三个相似。当在 S2 细胞中表达时，三个克隆基因产物中的每一个都被整合到与 PSE 功能性结合的蛋白质复合物中。我们还发现，上述构象差异对于 DmPBP45 尤其明显，本文将其鉴定为人 SNAP43 的直系同源物。 DmPBP45 与 U1 PSE 下游 DNA 螺旋两圈的 DNA 发生强烈交联，但仅与 U6 PSE 下游螺旋的半圈发生强烈交联。交联模式的这些实质性差异与 DmPBP-DNA 复合物的构象差异导致选择性 RNA 聚合酶招募到 U1 和 U6 启动子的模型一致。
Transcription of snRNA genes by either RNA polymerase 11 (U1 to U5) or RNA polymerase III (U6) is dependent upon a proximal sequence element (PSE) located approximately 40 to 60 bp upstream of the transcription start site. In Drosophila melanogaster, RNA polymerase specificity is determined by as few as three nucleotide differences within the otherwise well-conserved 21-bp PSE. Previous photo-cross-linking studies revealed that the D. melanogaster PSE-binding protein, DmPBP, contains three subunits (DmPBP45, DmPBP49, and DmPBP95) that associate with the DNA to form complexes that are conformationally distinct depending upon whether the protein is bound to a U1 or a U6 PSE. We have identified and cloned the genes that code for these subunits of DmPBP by virtue of their similarity to three of the five subunits of SNAP., the human PBP. When expressed in S2 cells, each of the three cloned gene products is incorporated into a protein complex that functionally binds to a PSE. We also find that the conformational difference referred to above is particularly pronounced for DmPBP45, herein identified as the ortholog of human SNAP43. DmPBP45 cross-linked strongly to DNA for two turns of the DNA helix downstream of the U1 PSE, but it cross-linked strongly for only a half turn of the helix downstream of a U6 PSE. These substantial differences in the cross-linking pattern are consistent with those of a model in which conformational differences in DmPBP-DNA complexes lead to selective RNA polymerase recruitment to U1 and U6 promoters.