Selection of Modified Ribosomes Using Novel Puromycins

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，以使体外制备含有多种非天然氨基酸（除α - l氨基酸外）的蛋白质，其数量足以用于常规生化研究。这将采用一种新的选择策略来完成，该策略使用修饰的嘌呤霉素衍生物，嘌呤霉素是氨基酰基trna的3'端结构模拟物，终止原核生物中（细胞）蛋白质的合成，导致细胞死亡。通过筛选一个大型的修饰核糖体文库，每个修饰核糖体都是在大肠杆菌中用（修饰的）质粒携带的23S核糖体RNA进行细胞转化后表达的，对含有特定结构修饰的氨基酸的嘌呤霉素类似物的敏感性，有可能鉴定出能够识别（并结合）这些附着在转移RNA上的相同氨基酸的核糖体。以这种方式增强了β氨基酸的掺入。在要求支持的5年期间，核糖体的23S rRNA成分将被改变，以允许将几种天然核糖体无法结合的修饰氨基酸结合到蛋白质中。这包括多种氨基酸，这将允许定义的特定位置/取代基的立体化学耐受的修饰核糖体。还将研究-氨基酸稳定蛋白质二级结构的能力。

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.