Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding.
Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding.
复制标题
P/P-和 P/E-位点 tRNAPhe 结合诱导大肠杆菌 70S 核糖体中 16S rRNA 的构象变化。
DOI：
10.1021/bi035369q
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Wollenzien,Paul
中科院分区：
文献类型：
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作者：
Noah,JamesW;Shapkina,TatjanaG;Nanda,Kavita;Huggins,Wayne;Wollenzien,Paul
使用UV交联研究了P/P-和P/E-位点tRNAPhe结合对大肠杆菌70S核糖体中16S rRNA结构的影响。在存在 deacyl-tRNAPheorN-乙酰基-Phe-tRNAPhe 使用 Poly(U) 或含有单个 Phe 密码子的 mRNA 类似物的情况下测定 16S rRNA 分子内交联的身份和频率。对于具有 Poly(U) 或 mRNA 类似物的 N-乙酰基-Phe-tRNAPhe，16S rRNA 中分子内交联 C967 × C1400 的频率与 tRNA 的结合化学计量成比例降低。对于带有聚（U）的脱酰基-tRNAPhe 也存在比例效应，但 C967 × C1400 频率的降低小于与 mRNA 类似物的 tRNA 结合化学计量。在含有或不含多胺的缓冲液中，C967 × C1400 交联的抑制作用相似。 RNA测序证实C967与C1400独家参与交联。一种分子间交联，即 16S rRNA (C1400) 至 tRNAPhe(U33)，是用 Poly(U) 或 mRNA 类似物制成的。这些结果表明，在 tRNA P 位点结合对所用 mRNA 类型敏感的过程中，C967 和 C1400 周围的小亚基发生了有限的结构变化。 70S 核糖体与 tRNA 复合物中不存在 C967 × C1400 交联，这与含有 tRNA 或 tRNA 类似物的 30S 和 70S 晶体结构一致；交联的出现表明空核糖体中该区域存在替代排列。
The effects of P/P- and P/E-site tRNAPhebinding on the 16S rRNA structure in theEscherichia coli70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNAPheorN-acetyl-Phe-tRNAPheusing poly(U) or an mRNA analogue containing a single Phe codon. ForN-acetyl-Phe-tRNAPhewith either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 × C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNAPhewith poly(U), but the decrease in the C967 × C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 × C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNAPhe(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 × C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.