Selection of Modified Ribosomes Using Novel Puromycins

使用新型嘌呤霉素选择修饰核糖体

• 批准号: 8733730
• 负责人: Sidney M. Hecht
• 金额: $ 19.37万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-13 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8733730
• 关键词: Aging; Amino Acids; Amino Acyl Transfer RNA; Amino Acyl-tRNA Synthetases; Architecture; Bacterial Proteins; Biochemical; Biocompatible Materials; Biological; Biological Assay; Cell Death; Cells; Chemistry; Codon Nucleotides; Communities; Development; Dipeptides; Disease; Electron Transport; Escherichia coli; Exhibits; Genetic Code; Goals; In Vitro; Investigation; Libraries; Ligand Binding; Malignant Neoplasms; Mediating; Messenger RNA; Modification; Molecular; Nature; Organism; Peptides; Peptidyltransferase; Plasmids; Positioning Attribute; Predisposition; Preparation; Production; Prokaryotic Cells; Property; Protein Biosynthesis; Proteins; Proteolysis; Publishing; Puromycin; RNA; RNA, Ribosomal, 23S; Research; Ribosomal RNA; Ribosomes; Roentgen Rays; Shapes; Structure; System; Techniques; Time; Transfer RNA; Vertebral column; analog; cell transformation; improved; in vivo; innovation; insight; interest; method development; novel; peptidomimetics; public health relevance; screening; stereochemistry; synthetic biology

DESCRIPTION (provided by applicant): The long term goals of this research include altering the 23S RNA in ribosomes to enable the in vitro preparation of proteins containing a broad variety of unnatural amino acids other than alpha-L-amino acids in quantities sufficient for routine biochemical studies. This will be done employing a novel selection strategy that uses modified derivatives of puromycin, a structural mimic of the 3'-end of aminoacyl-tRNA which terminates (cellular) protein synthesis in prokaryotes, resulting in cell death. By screening a large library of modified ribosomes, each of which has been expressed in E. coli following cell transformation with a (modified) plasmid-born 23S ribosomal RNA, for sensitivity to puromycin analogues containing a specific, structurally modified amino acid, it is possible to identify ribosomes potentially capable of recognizing (and incorporating) those same amino acids attached to transfer RNAs. The enhanced incorporation of beta amino acids has been achieved in this fashion. For the five-year period of requested support, the 23S rRNA constituent of ribosomes will be altered to permit the incorporation into proteins of several types of modified amino acids that native ribosomes cannot incorporate. This includes a variety of beta amino acids, which will allow definition of the specific positions/stereochemistry of substituents tolerated by the modified ribosomes. Also studied will be the ability of the beta amino acids to stabilize secondary structures in proteins.

描述（由申请人提供）：本研究的长期目标包括改变核糖体中的 23S RNA，以能够体外制备含有除 α-L-氨基酸之外的多种非天然氨基酸的蛋白质，其数量足以满足常规需求生化研究。这将通过采用一种新的选择策略来完成，该策略使用嘌呤霉素的修饰衍生物，嘌呤霉素是氨酰基-tRNA 3'端的结构模拟物，可终止原核生物中的（细胞）蛋白质合成，导致细胞死亡。通过筛选大型修饰核糖体文库（每个修饰核糖体文库均在使用（修饰的）质粒生成的 23S 核糖体 RNA 进行细胞转化后在大肠杆菌中表达，以检测对含有特定结构修饰氨基酸的嘌呤霉素类似物的敏感性，可能鉴定出能够识别（并整合）附着在转移 RNA 上的相同氨基酸的核糖体。以这种方式实现了β氨基酸的增强掺入。在请求支持的五年期限内，核糖体的 23S rRNA 成分将被改变，以允许将天然核糖体无法掺入的几种类型的修饰氨基酸掺入蛋白质中。这包括各种β氨基酸，这将允许定义修饰核糖体耐受的取代基的特定位置/立体化学。还研究了β氨基酸稳定蛋白质二级结构的能力。