Selection of Modified Ribosomes Using Novel Puromycins

描述(申请人提供)：这项研究的长期目标包括改变核糖体中的23S核糖核酸，使体外制备的蛋白质含有大量非天然氨基酸，而不是α-L-氨基酸，其数量足以进行常规生化研究。这将使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，它是氨基酰-tRNA的3‘端的结构模拟，它终止原核生物(细胞)蛋白质的合成，导致细胞死亡。通过筛选修饰的核糖体的大型文库，每个修饰的核糖体都在(修改的)质粒出生的23S核糖体RNA的细胞转化后在大肠杆菌中表达，以检测对含有特定的结构修饰的氨基酸的扑霉素类似物的敏感性，有可能识别潜在地能够识别(并结合)与转移RNA连接的那些相同的氨基酸的核糖体。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间，核糖体的23S rRNA成分将被改变，以允许将天然核糖体无法结合的几种修饰氨基酸并入蛋白质中。这包括各种β氨基酸，这将允许定义被修饰的核糖体所容忍的取代基的特定位置/立体化学。还将研究β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.