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：
7061644
负责人：
SANTIAGO MARTINEZ-CALVILLO
金额：
$ 5.27万
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/7061644
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（动力学阶层），导致疾病范围从无症状到致死，导致广泛的人类苦难和死亡。 LMJF提出了基因表达的非典型机制，因为转录似乎仅在每个染色体的几个区域中启动，从而产生长的多重传递转录本，这些转录物是通过移植以产生成熟的mRNA来处理的。关于RNA聚合酶III（POL III）的转录的动力学塑料，几乎不知所知，该RNA聚合酶III（POLII III）在细胞代谢中转录了几种保守和丰富的小RNA（例如TRNA，5S rRNA和SNRNA）。我们的主要目的是表征利什曼尼亚主要弗里德林（LMJF）中的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（和其他动力质体）中的Pol III启动子和转录因子与其他真核生物上存在的启动子和转录因子有很大差异。具体目的是： 1。分析LMJF中Pol III基因的转录。转录启动位点将由5'race映射，并用poly（a）尾的RNA定位的RT-PCR终止位点。将使用跨越编码和基因间区域的单链DNA片段进行核跑步分析。启动子活性将通过瞬时转染研究测试。 2。检查Pol III启动子与转录机械之间的DNA蛋白相互作用。 DNA蛋白相互作用将通过电泳迁移率转移测定法分析。将进行DNase I足迹测定，以识别与Pol III复合物相互作用的特定DNA序列。 3。确定LMJF中POL III复合物的蛋白质成分。串联亲和力纯化（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来鉴定。