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23 S RNA，以能够体外制备含有大量非天然氨基酸（α-L-氨基酸除外）的蛋白质，其量足以进行常规生化研究。这将采用一种新的选择策略来完成，该策略使用嘌呤霉素的修饰衍生物，嘌呤霉素是氨酰-tRNA的3 '-末端的结构模拟物，其终止原核生物中的（细胞）蛋白质合成，导致细胞死亡。通过筛选一个大的修饰核糖体文库，每个修饰核糖体都已在大肠杆菌中表达。在用（修饰的）质粒产生的23 S核糖体RNA转化大肠杆菌细胞后，为了对含有特定的、结构修饰的氨基酸的嘌呤霉素类似物敏感，有可能鉴定出潜在地能够识别（和掺入）那些连接到转移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。在所要求的五年支持期内，核糖体的23 S rRNA成分将被改变，以允许将天然核糖体不能掺入的几种类型的修饰氨基酸掺入蛋白质中。这包括多种β氨基酸，其将允许限定被修饰的核糖体耐受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.