TRNA PROCESSING NUCLEASES FROM LIVER MITOCHONDRIA

TRNA 处理来自肝线粒体的核酸酶

• 批准号: 3294606
• 负责人: GLENN C VAN TUYLE
• 金额: $ 8.13万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-29 至 1991-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3294606
• 关键词: DNA directed RNA polymerase; RNA; chemical fingerprinting; cytoplasm; enzyme structure; enzyme substrate; gel electrophoresis; gel filtration chromatography; genetic promoter element; genetic transcription; high performance liquid chromatography; laboratory rat; liver metabolism; mitochondria; mitochondrial DNA; molecular size; nuclease; nucleic acid sequence; nucleic acid structure; transfer RNA

The 5' and 3' tRNA processing nucleases from liver mitochondria will be further purified using current methods of enzyme purification. The molecular sizes of the native forms of these two processing nucleases will be measured by sedimentation analysis and moleuclar sieving techniques, and the multiplicity of protein components will be determined by SDS-polyacrylamide gel electrophoresis. Functional studies of the highly purified enzymes will be performed using normal and altered mitochondrial (32P) pre-tRNA transcripts produced in vitro from mtDNA fragments cloned into transcription vectors, or from synthetic templates containing the T7 RNA polymerase promoter. The optimal reaction conditions and spectrum of substrates will be determined using normal mitochondrial tRNA precursor transcripts, and the sequence/structural features of precursor substrates that are critical to recognition and efficient processing will be examined with altered pre-tRNA transcripts produced from chemically synthesized templates. Processed (32P) products will be separated on polyacrylamide/urea gels, excised, and analyzed by conventional fingerprinting using primary and secondary digestions, and end-group analyses. The activity of the 3' processing nuclease will be assayed with (32P) 5'- processed tRNA intermediates. It will be determined whether the mitochondrial 3' processing nuclease is a precise endonuclease, consistent with the activity of the 5' enzyme. The existence of an essential RNA component associated with the 5' and 3' processing nucleases will be tested by sensitivity of these enzymes to micrococcal nuclease digestion. The RNA component(s) will be isolated and sequenced by current RNA sequencing proteocols to determine the probable site of origin (mitochondrial or nuclear) of the RNA(s). In addition, the cytosolic (extra-mitochondrial) 5' processing nuclease will be further purified for direct comparison with the mitochondrial counterpart.

来自肝脏线粒体的5‘和3’ tRNA加工核酸酶