RNA Polymerase III transcription in Leishmania major

大型利什曼原虫中的 RNA 聚合酶 III 转录

基本信息

批准号：
6943808
负责人：
SANTIAGO MARTINEZ-CALVILLO
金额：
$ 5.4万
依托单位：
UNIVERSIDAD NACIONAL AUTONOMA DE MEXICO
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-05-01 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6943808
关键词：
DNA directed RNA polymerase DNA footprinting Leishmania major Mexico affinity chromatography cell line gel mobility shift assay gene expression genetic mapping genetic promoter element genetic transcription intermolecular interaction mass spectrometry nuclear runoff assay nucleic acid sequence protein purification transcription factor

项目摘要

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.

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 呈现出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起始，产生长的多顺反子转录物，并通过转拼处理产生成熟的 mRNA。我们对动质体中 RNA 聚合酶 III (Pol III) 的转录知之甚少，RNA 聚合酶 III 转录多种保守且丰富的小 RNA（例如 tRNA、5S rRNA 和 snRNA），这些小 RNA 在细胞代谢中发挥着关键作用。我们的主要目标是表征大型利什曼原虫 Friedlin (LmjF) 的 Pol III 启动子和转录复合物，其基因组序列最近已完成。具体的假设是，LmjF（和其他动质体）中的 Pol III 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。转录起始位点将通过 5'-RACE 进行定位，终止位点则通过 RT-PCR 使用聚 (A) 尾 RNA 进行定位。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分析进行分析。将进行 DNase I 足迹分析，以确定与 Pol III 复合物相互作用的特定 DNA 序列。 3. 鉴定 LmjF 中 Pol III 复合物的蛋白质成分。串联亲和纯化（TAP-tag）方法将用于纯化 Pol III 转录复合物。这些复合物中的蛋白质成分将通过质谱分析进行鉴定。