Vankan P, Filipowicz W
Friedrich Miescher-Institut, Basel, Switzerland.
EMBO J. 1989 Dec 1;8(12):3875-82. doi: 10.1002/j.1460-2075.1989.tb08566.x.
The U2 and U5 snRNA genes of Arabidopsis thaliana contain in their promoter regions two elements with conserved sequence and position. To test the significance of this conservation we have made a construction in which the promoter of the U2 RNA gene is replaced by the synthetic 98 bp long sequence containing the two conserved elements: an upstream sequence element, GTCCCACATCG (USE, pos. -78 to -68), and a TATA-like sequence TATAAATA (-33 to -26), positioned approximately three helical turns apart, as in the wild-type promoter. This synthetic promoter efficiently drove transcription of the U2 gene in transfected protoplasts of Nicotiana plumbaginifolia. The importance of the individual elements and of their position within the promoter was investigated. Deletion of the USE, change of its orientation, and some single point mutations all decreased transcription 10- to 20-fold, and replacement of the TATA-like element by an unrelated sequence inactivated the promoter. Mutants in which the spacing between the USE and TATAAATA was changed were less active but no correlation was observed between promoter activity and insertion of either odd or even numbers of half helical turns. Insertion of a spacer between TATAAATA and the cap site resulted in accumulation of U2 RNA with an extended 5' end, indicating that the TATAAATA element is responsible for selection of the initiation site. The data indicate that the promoters of RNA polymerase II-specific U-snRNA genes in higher plants differ from their animal counter-parts and also from plant mRNA gene promoters. They contain two essential elements, an USE, an element found only in U-snRNA genes, and a TATA element which is indistinguishable from the TATA boxes of mRNA-coding genes.
拟南芥的U2和U5小核RNA基因在其启动子区域含有两个序列和位置保守的元件。为了测试这种保守性的意义，我们构建了一个结构，其中U2 RNA基因的启动子被一个合成的98 bp长序列取代，该序列包含两个保守元件：一个上游序列元件GTCCCACATCG（USE，位置-78至-68）和一个类TATA序列TATAAATA（-33至-26），它们的位置大约相隔三个螺旋圈，就像在野生型启动子中一样。这个合成启动子在转染的烟草叶肉原生质体中有效地驱动了U2基因的转录。我们研究了各个元件及其在启动子内位置的重要性。删除USE、改变其方向以及一些单点突变均使转录降低了10至20倍，用无关序列取代类TATA元件使启动子失活。改变USE和TATAAATA之间间距的突变体活性较低，但未观察到启动子活性与奇数或偶数个半螺旋圈插入之间的相关性。在TATAAATA和帽位点之间插入一个间隔序列导致5'端延长的U2 RNA积累，表明TATAAATA元件负责起始位点的选择。数据表明，高等植物中RNA聚合酶II特异性U - snRNA基因的启动子与其动物对应物不同，也与植物mRNA基因启动子不同。它们包含两个必需元件，一个USE，这是仅在U - snRNA基因中发现的元件，以及一个与mRNA编码基因的TATA框无法区分的TATA元件。
