Mechanisms of transcription initiation in mitochondria

线粒体转录起始机制

• 批准号: 9257439
• 负责人: Dmitry Temiakov
• 金额: $ 6.96万
• 依托单位: ROWAN UNIVERSITY SCHOOL/OSTEOPATHIC MED
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-14 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9257439
• 关键词: Active Sites; Aging; Amino Acids; Bacteriophage T7; Binding; Biochemical; Biological Assay; Blindness; Cells; Chloroplasts; Circular DNA; Complex; Crystallization; DNA; DNA Polymerase I; DNA Polymerase II; DNA Polymerase III; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Defect; Degenerative Disorder; Development; Disease; Distant; Enzymes; Epitopes; Family; Gene Expression; Genes; Genetic Transcription; Goals; Human; Human Development; Impairment; In Vitro; Knowledge; Lactic Acidosis; Leg; MERRF Syndrome; Maintenance; Malignant Neoplasms; Maps; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Encephalomyopathies; Mitochondrial RNA; Modeling; Molecular; Mutagenesis; Myoclonic Epilepsies; Myopathy; Nature; Nuclear; Organelles; Oxidative Phosphorylation; Parasites; Pathology; Peptide Initiation Factors; Protein Subunits; Proteins; RNA Transport; Recruitment Activity; Regulation; Resolution; Ribosomes; Role; Specificity; Stroke; Structure; Syndrome; System; Testing; Transcription Initiation; Transcription Initiation Site; Work; age related; crosslink; experimental study; hearing impairment; melting; mitochondrial genome; novel; novel therapeutics; promoter; public health relevance; senescence; targeted treatment; therapeutic target; therapy development; transcription factor

DESCRIPTION (provided by applicant): Human mitochondria maintain a small circular DNA that encodes several subunits of the proteins involved in oxidative phosphorylation as well as ribosomal and transport RNA. Defects in mtDNA expression result in a number of myopathies such as MELAS (mitochondrial encephalomyopath, lactic acidosis, and stroke-like syndrome), MERRF (myoclonic epilepsy with ragged red fibers syndrome), hearing and vision loss, and others. Moreover, mitochondria are implicated in age-related pathology, senescence and cancer and thus represent a rational but understudied therapeutic target. The key enzyme involved in mtDNA expression, mitochondrial RNA polymerase (mtRNAP) belongs to a family of single-subunit RNAPs that is distinct from the multi-subunit cellular RNAPs but distantly related to RNAPs of bacteriophage T7, the pol I familty of DNA polymerases, and single-subunit RNAPs from chloroplasts. However, unlike T7 RNAP, human mtRNAP requires two transcription factors, TFAM and TFB2M for efficient initiation, and its transcription appears to be regulated by a number of transcription factors. Studies of the structure and function of the mtRNAP and molecular mechanisms of its transcription are important for understanding the regulation of mitochondrial genome expression. This, in turn, will determine our ability to influence various mitochondrial functions and as a consequence, to treat mitochondria-associated diseases. The goal of this project is to determine the molecular mechanisms of transcription initiation by human mtRNAP. The specific aims are as follows: Aim 1. Determine the molecular basis of transcription initiation by mtRNAP. The high resolution structure of mtRNAP will be used to guide biochemical experiments to probe the function of mtRNAP domains that are involved in promoter binding, recognition, melting and interactions with transcription factors. Transitions of mtRNAP during initiation to elongation will be probed by cross-linking and by structural analysis of the mtRNAP elongation complex. Aim 2. Determine function and structure of the pre-initiation complex. Using protein-protein cross-linking we will determine the organization of a novel transcription intermediate - pre-initiation complex formed with TFAM and mtRNAP on promoter DNA. Interacting regions in both TFAM and mtRNAP will be mapped and their functional importance probed by mutagenesis. The structure of the pre-initiation complex will be determined at the atomic resolution and its function probed in various biochemical assays. Aim 3. Determine function and structure of the open promoter complex. Protein-protein cross-linking using artificial photo reactive amino acid will be used to test the model of TFB2M interaction with mtRNAP during initiation and to probe assembly of the initiation complex. The role of TFB2M in transcription start site selection, promoter specificity and promoter melting will be examined. The crystal structure of the core initiation complex that includes mtRNAP, TFAM, TFB2M and promoter DNA will be determined.

描述（由申请人提供）： 人线粒体维持一个小的圆形DNA，该DNA编码参与氧化磷酸化以及核糖体和转运RNA的蛋白质的几个亚基。 mtDNA表达的缺陷导致许多肌病，例如Melas（线粒体脑疗法，乳酸酸中毒和中风样综合征），MERRF（肌钙发癫痫患有破烂的红色纤维综合征），听力和视力丧失等。 此外，线粒体与年龄相关的病理学，衰老和癌症有关，因此代表了一个理性但正在研究的治疗靶点。线粒体RNA聚合酶（MTRNAP）涉及MTDNA表达的关键酶属于一个单一亚基RNAP家族，与多含量的细胞RNAP不同，但与细菌噬菌体T7的rnap相关，但与T7的RNAP相关，DNA Polymerass of DNA Polymerass s和Singlemubunit rnaps rnaps rnaps rnaps rnaps rnaps rnaps rnaps rnaps rnaps rnaps rnaps rnaps rnaps。但是，与T7 RNAP不同，人类MTRNAP需要两个转录因子，即TFAM和TFB2M才能有效启动，并且其转录似乎受许多转录因子的调节。对MTRNAP的结构和功能的研究和其转录的分子机制对于理解线粒体基因组表达的调节很重要。反过来，这将决定我们影响各种线粒体功能的能力，并因此治疗与线粒体相关疾病的能力。该项目的目的是确定人类MTRNAP转录启动的分子机制。具体目的如下：目标1。确定转录启动的分子基础 由mtrnap。 MTRNAP的高分辨率结构将用于指导生化实验，以探测与启动子结合，识别，熔融，熔化和与转录因子相互作用的MTRNAP结构域的功能。 MTRNAP在启动伸长过程中的过渡将通过交联和MTRNAP伸长络合物的结构分析来探测。 AIM 2。确定启动前复合物的功能和结构。使用蛋白质 - 蛋白质交联，我们将确定由TFAM和MTRNAP在启动子DNA上形成的新型转录中间体 - 发射复合物的组织。 TFAM和MTRNAP中的相互作用区域将被映射，并通过诱变探测其功能重要性。预生效复合物的结构将在原子分辨率及其功能中确定，并在各种生化测定中探测。 AIM 3。确定开放启动子复合物的功能和结构。使用人工照片反应性氨基酸的蛋白质 - 蛋白质交联将用于测试启动过程中与MTRNAP相互作用的模型，并探测起始复合物的组装。将检查TFB2M在转录起始位点选择，启动子特异性和启动子熔化中的作用。将确定包括MTRNAP，TFAM，TFB2M和启动子DNA的核心起始复合物的晶体结构。