核糖体A位点中23S rRNA与tRNA之间的碱基配对。

Base-pairing between 23S rRNA and tRNA in the ribosomal A site.

作者信息

Kim D F, Green R

机构信息

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

出版信息

Mol Cell. 1999 Nov;4(5):859-64. doi: 10.1016/s1097-2765(00)80395-0.

DOI:10.1016/s1097-2765(00)80395-0

PMID:10619032

Abstract

The aminoacyl (A site) tRNA analog 4-thio-dT-p-C-p-puromycin (s4TCPm) photochemically cross-links with high efficiency and specificity to G2553 of 23S rRNA and is peptidyl transferase reactive in its cross-linked state, establishing proximity between the highly conserved 2555 loop in domain V of 23S rRNA and the universally conserved CCA end of tRNA. To test for base-pairing interactions between 23S rRNA and aminoacyl tRNA, site-directed mutations were made at the universally conserved nucleotides U2552 and G2553 of 23S rRNA in both E. coli and B. stearothermophilus ribosomal RNA and incorporated into ribosomes. Mutations at G2553 resulted in dominant growth defects in E. coli and in decreased levels of peptidyl transferase activity in vitro. Genetic analysis in vitro of U2552 and G2553 mutant ribosomes and CCA end mutant tRNA substrates identified a base-pairing interaction between C75 of aminoacyl tRNA and G2553 of 23S rRNA.

摘要

氨酰基（A位点）tRNA类似物4-硫代-dT-p-C-嘌呤霉素（s4TCPm）以高效且特异的方式与23S rRNA的G2553发生光化学交联，并且在其交联状态下具有肽基转移酶活性，这表明23S rRNA结构域V中高度保守的2555环与tRNA普遍保守的CCA末端之间存在紧密联系。为了检测23S rRNA与氨酰基tRNA之间的碱基配对相互作用，在大肠杆菌和嗜热栖热菌核糖体RNA的23S rRNA普遍保守核苷酸U2552和G2553处进行了定点突变，并将其整合到核糖体中。G2553处的突变导致大肠杆菌中出现显性生长缺陷，并且体外肽基转移酶活性水平降低。对U2552和G2553突变核糖体以及CCA末端突变tRNA底物进行的体外遗传分析确定了氨酰基tRNA的C75与23S rRNA的G2553之间存在碱基配对相互作用。