Transcription termination and 3'-End processing of the spliced leader RNA in kinetoplastids.
Transcription termination and 3'-End processing of the spliced leader RNA in kinetoplastids.
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动质体中剪接前导 RNA 的转录终止和 3 端加工。
DOI：
10.1128/mcb.19.2.1595
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发表时间：
1999
影响因子：
5.3
通讯作者：
Campbell,DA
中科院分区：
文献类型：
--
作者：
Sturm,NR;Yu,MC;Campbell,DA
通过反式剪接增加一个39个核苷酸的剪接前导序列（SL）是所有锥虫核mRNA的基本要求。蜥蜴利什曼原虫的SL RNA是由一种聚合酶合成的96-nt前体转录物，该聚合酶与聚合酶II最相似。为了分析SL RNA的发生，我们突变SL RNA内含子的结构和序列元素：茎环II和III，Sm结合位点，和下游的T道。使用外显子标记的SL RNA基因，我们研究了由第二位点10-bp接头扫描诱变系列和定向诱变产生的表型。在这里，我们报告，转录终止的T道，这是共同的3′端的所有动质体SL RNA基因，并在体内有效终止需要超过6个T的。我们描述的突变体的SL RNA结束在T道或似乎缺乏有效的终止，但可以产生野生型3′端。转录活性核提取物显示T道中的交错产物，由八个或更多个T指导。体内和体外实验结果表明，SL RNA转录终止在T束中是交错的，随后是溶核加工，产生成熟的3′端。我们发现Sm结合位点和茎环III结构是正确的3′-末端形成所必需的。因此，我们已经确定了SL RNA基因的转录终止元件。其终止机制不同于脊椎动物小核RNA基因和蛔虫SL RNA同源物。
Addition of a 39-nucleotide (nt) spliced leader (SL) bytranssplicing is a basic requirement for all trypanosome nuclear mRNAs. The SL RNA inLeishmania tarentolaeis a 96-nt precursor transcript synthesized by a polymerase that resembles polymerase II most closely. To analyze SL RNA genesis, we mutated SL RNA intron structures and sequence elements: stem-loops II and III, the Sm-binding site, and the downstream T tract. Using an exon-tagged SL RNA gene, we examined the phenotypes produced by a second-site 10-bp linker scan mutagenic series and directed mutagenesis. Here we report that transcription is terminated by the T tract, which is common to the 3′ end of all kinetoplastid SL RNA genes, and that more than six T’s are required for efficient termination in vivo. We describe mutants whose SL RNAs end in the T tract or appear to lack efficient termination but can generate wild-type 3′ ends. Transcriptionally active nuclear extracts show staggered products in the T tract, directed by eight or more T’s. The in vivo and in vitro data suggest that SL RNA transcription termination is staggered in the T tract and is followed by nucleolytic processing to generate the mature 3′ end. We show that the Sm-binding site and stem-loop III structures are necessary for correct 3′-end formation. Thus, we have defined the transcription termination element for the SL RNA gene. The termination mechanism differs from that of vertebrate small nuclear RNA genes and the SL RNA homologue inAscaris.