Base-pairing between 23S rRNA and tRNA in the ribosomal A site
Base-pairing between 23S rRNA and tRNA in the ribosomal A site
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DOI：
10.1016/s1097-2765(00)80395-0
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发表时间：
1999-11-01
期刊：
影响因子：
16
通讯作者：
Green, R
中科院分区：
文献类型：
--
作者：
Kim, DF;Green, R
氨基酰(A位)tRNA类似物4-硫代-dt-p-C-p-嘌呤霉素(S(4)TCPM)通过光化学方法高效、特异地与23S rRNA的G2553发生交联，并在其交联状态下具有肽基转移酶活性，在23S rRNA的V区高度保守的2555环与tRNA普遍保守的CCA端之间建立了接近的位置。为了检测23S rRNA和氨基酰tRNA之间的碱基配对相互作用，在E、Coli和B.stearthermophilus核糖体RNA中对23S rRNA中普遍保守的核苷酸U2552和G2553进行了定点突变，并将其整合到核糖体中。在体外，G2553突变导致了大肠杆菌的显性生长缺陷和肽基转移酶活性水平的降低。U2552和G2553突变核糖体和CCA末端突变tRNA底物的体外遗传分析表明，氨基酰tRNA的C75和23S rRNA的G2553之间存在碱基配对相互作用。
The aminoacyl (A site) tRNA analog 4-thio-dT-p-C-p-puromycin (s(4)TCPm) 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.