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(动塑形目)，可引起从无症状到致命的一系列疾病，导致广泛的人类痛苦和死亡。LmjF提出了非典型的基因表达机制，因为转录似乎只在每条染色体的几个区域开始，产生长的多顺反子转录本，通过反式剪接处理产生成熟的mRNAs。在动体中，关于RNA聚合酶III(Pol III)的转录作用知之甚少，它转录几个保守而丰富的小RNA(如tRNAs、5S rRNAs和SnRNAs)，这些小RNA在细胞代谢中发挥关键作用。我们的主要目标是鉴定最近完成基因组测序的大利什曼原虫(LmjF)的POLIII启动子和转录复合体。具体的假设是LmjF(和其他动质粒)中的POLIII启动子和转录因子与其他真核生物中的启动子和转录因子有很大不同。具体目标是： 1.分析LmjF中Pol III基因的转录。转录起始点将通过5‘-RACE定位，终止位点将通过RT-PCR与Poly(A)-Tail RNA定位。核连续分析将用跨越编码区和基因间隔区的单链DNA片段进行。启动子活性将通过瞬时转基因研究进行测试。 2.研究Pol III启动子与转录机制之间的DNA-蛋白质相互作用。DNA-蛋白质的相互作用将通过电泳迁移率变化分析来分析。将进行DNase I足迹分析，以确定与Pol III复合体相互作用的特定DNA序列。 3.鉴定LmjF中Pol III复合体的蛋白质组分。串联亲和纯化(TAP-Tag)方法将用于Pol III转录复合体的纯化。这些复合体中的蛋白质成分将通过质谱分析进行鉴定。

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.