Transcription of U6 Small Nuclear RNA

人类核U6 RNA可能是由聚合酶III转录的，而不同于其他参与前mRNA剪接的丰富的SnRNAs。然而，来自多种生物的U6基因的5‘侧翼区包含的转录调控元件似乎与PolII转录的U1和U2基因非常相似。此外，与典型的polIII基因不同的是，在U6SnRNA基因中没有发现内控元件。在一个项目中，将对人类U6基因近端区域的转录控制元件进行详细分析。利用体外转录和体内实验研究导致U6基因转录的启动子元件(S)。位于5‘端的U6“基本”启动子似乎受到严格的限制。控制元素的相对间距和/或方向。将通过构建一系列U6基因启动子的缺失和插入突变体来详细研究间距的作用。U6转录的机制也将通过部分纯化反式作用因子来研究。支持准确U6转录的S100提取物将通过几个层析步骤进行分级。直接转录分析和DNA-蛋白质结合分析，如DNA酶足迹分析，将被用来鉴定部分纯化的因子。这一应用的中心焦点是控制人类U6小核RNA(SnRNA)的转录。这个SnRNA是前信使RNA剪接的辅助因子，这是真核细胞基因表达的基本步骤。

Human nuclear U6 RNA is presumably tanscribed by polymerase III unlike the other abundant snRNAs involved in pre-mRNA splicing. However the 5' flanking regions of U6 genes from a variety of organisms contain transcription control elements that appear to be very similar to those employed on pol II- transcribed U1 and U2 genes. Moreover, unlike typical pol III genes, no internal control elements have been found in the U6 snRNA gene. In one project a detailed analysis will be made of the transcription control elements in the proximal region of a human U6 gene. Experiments are proposed that use in vitro transcription and experession in vivo from transfected genes to investigate the promoter element(s) that cause the U6 gene to be transcribed by RNA polymerase III. The U6 "basal" promoter situated in the 5' proximal region appears to be rigidly constrained with respect to the relative spacing and/or orientation of control elements. The role of spacing will be studied in detail through the construction of a series of deletion and insertion mutants in the U6 gene promoter. The mechanism of U6 transcription will also be studied by partial purification of trans-acting factors. A S100 extract that supports accurate U6 transcription will fractionated through several chromatographic steps. Direct transcription assays and DNA- protein binding analyses such as DNase footprinting will be used to characterize the partially purified factors. The central focus of this application is the control of human U6 small nuclear RNA (snRNA) transcription. This snRNA is a cofactor in pre-messenger RNA splicing, a fundamental step of gene expression in eukaryotic cells.