RNA Polymerase III transcription in Leishmania major

描述（由申请人提供）： 利什曼原虫是一种寄生原生动物（动质体目），可引起一系列疾病，从无症状到致命，导致广泛的人类痛苦和死亡。LmjF呈现非典型的基因表达机制，因为转录似乎仅在每条染色体的几个区域中启动，产生长的多顺反子转录物，其通过反式剪接加工以产生成熟mRNA。RNA聚合酶III（Pol III）转录在动质体中起关键作用的几种保守和丰富的小RNA（如tRNA、5S rRNA和snRNA），其转录在细胞代谢中起关键作用。我们的主要目标是在利什曼原虫主要Friedlin（LmjF），其基因组序列已于最近完成的Pol III启动子和转录复合物的特点。具体的假设是，在LmjF（和其他动质体）的Pol III启动子和转录因子显着不同，从其他真核生物。具体目标是： 1.分析LmjF中Pol III基因的转录。转录起始位点将通过5 '-RACE定位，终止位点通过RT-PCR用poly（A）-tailed RNA定位。将使用跨越编码区和基因间区的单链DNA片段进行核连续分析。启动子活性将通过瞬时转染研究进行测试。 2.检查Pol III启动子和转录机制之间的DNA-蛋白质相互作用。DNA-蛋白质相互作用将通过电泳迁移率变动试验进行分析。将进行DNA酶I足迹分析，以鉴定与Pol III复合物相互作用的特定DNA序列。 3.鉴定LmjF中Pol III复合物的蛋白质组分。串联亲和纯化（TAP-标签）方法将用于纯化Pol III转录复合物。这些复合物中的蛋白质组分将通过质谱分析来鉴定。

DESCRIPTION (provided by applicant): Leishmania is a parasitic protozoan (order Kinetoplastida) that causes a spectrum of disease ranging from asymptomatic to lethal, resulting in widespread human suffering and death. LmjF presents atypical mechanisms of gene expression, since transcription seems to initiate in only a few regions per chromosome, generating long polycistronic transcripts that are processed by trans-splicing to produce mature mRNAs. Little is known in kinetoplastids about transcription by RNA Polymerase III (Pol III), which transcribes several conserved and abundant small RNAs (such as tRNAs, 5S rRNAs and snRNAs) that play critical roles in cell metabolism. Our main objective is to characterize Pol III promoters and transcriptional complexes in Leishmania major Friedlin (LmjF), whose genome sequence has been recently completed. The specific hypothesis is that Pol III promoters and transcription factors in LmjF (and other kinetoplastids) differ considerably from those present on other eukaryotes. The specific aims are to: 1. Analyze transcription of Pol III genes in LmjF. Transcription start sites will be mapped by 5'-RACE, and termination sites localized by RT-PCR with poly(A)-tailed RNA. Nuclear run-on analysis will be performed with single-stranded DNA fragments spanning coding and intergenic regions. Promoter activity will be tested by transient-transfection studies. 2. Examine the DNA-protein interactions between the Pol III promoters and the transcription machinery. DNA-protein interactions will be analyzed by electrophoretic mobility shift assays. DNase I footprinting assays will be carried out to identify the specific DNA sequences that interact with the Pol III complex. 3. Identify the protein components of the Pol III complex in LmjF. The tandem affinity purification (TAP-tag) method will be used to purify Pol III transcriptional complexes. The protein components in these complexes will be identified by mass-spectrometry analysis.