U6 SMALL NUCLEAR-RNA IS TRANSCRIBED BY RNA POLYMERASE-III
U6 SMALL NUCLEAR-RNA IS TRANSCRIBED BY RNA POLYMERASE-III
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DOI：
10.1073/pnas.83.22.8575
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发表时间：
1986-11-01
影响因子：
11.1
通讯作者：
PEDERSON, T
中科院分区：
文献类型：
--
作者：
KUNKEL, GR;MASER, RL;PEDERSON, T
从人类基因组文库中分离出与 U6 小核 RNA 同源的 DNA 片段并进行测序。 U6 DNA克隆的直接5''侧翼区域与潜在的小鼠U6基因具有显着的同源性，包括位于转录起始位点上游26-29个核苷酸位置的“TATA盒”。尽管该序列元件是RNA聚合酶II启动子的特征，但U6基因还包含聚合酶III“框A”基因内控制区和在3''末端（非编码链）的典型的五个胸腺嘧啶序列。人类 U6 DNA 克隆在缺乏聚合酶 II 活性的 HeLa 细胞 S100 提取物中准确转录。 S100提取物中的U6 RNA转录对1μg/ml的α-鹅膏蕈素具有抗性，但在200μg/ml的浓度下被完全抑制。体外转录本和体内合成的 U6 RNA 指纹的比较揭示了序列一致性。与U1和U2 RNA转录相比，分离的HeLa细胞核中的U6 RNA合成也表现出对α-鹅膏蕈素的低敏感性，U1和U2 RNA转录在1μg/ml下被抑制>90％。此外，在分离的细胞核中合成的 U6 RNA 可以被针对 La 抗原的抗体有效地免疫沉淀，La 抗原是一种已知与大多数其他 RNA 聚合酶 III 转录物结合的蛋白质。这些结果表明，与 U1-U5 小核 RNA 的哺乳动物基因的聚合酶 II 指导转录相反，人 U6 RNA 由 RNA 聚合酶 III 转录。
A DNA fragment homologous to U6 small nuclear RNA was isolated from a human genomic library and sequenced. The immediate 5''-flanking region of the U6 DNA clone had significant homology with a potential mouse U6 gene, including a "TATA box" at a position 26-29 nucleotides upstream from the transcription start site. Although this sequence element is characteristic of RNA polymerase II promoters, the U6 gene also contained a polymerase III "box A" intragenic control region and a typical run of five thymines at the 3'' terminus (noncoding strand). The human U6 DNA clone was accurately transcribed in a HeLa cell S100 extract lacking polymerase II activity. U6 RNA transcription in the S100 extract was resistant to .alpha.-amanitin at 1 .mu.g/ml but was completely inhibited at 200 .mu.g/ml. A comparison of fingerprints of the in vitro transcript and of U6 RNA synthesized in vivo revealed sequence congruence. U6 RNA synthesis in isolated HeLa cell nuclei also displayed low sensitivity to .alpha.-amanitin, in contrast to U1 and U2 RNA transcription, which was inhibited > 90% at 1 .mu.g/ml. In addition, U6 RNA synthesized in isolated nuclei was efficiently immunoprecipitated by an antibody against the La antigen, a protein known to bind most other RNA polymerase III transcripts. These results establish that, in contrast to the polymerase II-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase III.