Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA
Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA
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DOI：
10.1006/jmbi.2001.4612
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发表时间：
2001-05-18
影响因子：
5.6
通讯作者：
Uhlenbeck, OC
中科院分区：
文献类型：
--
作者：
Pleiss, JA;Uhlenbeck, OC
使用一组45种不同的tRNA（每种含有覆盖分子上半部分的单个脱氧核苷酸取代）与高通量核糖核酸酶保护测定结合，以研究2'羟基在稳定来自嗜热栖热菌的延伸因子Tu（EF-Tu）的复合物中的热力学作用。鉴定了5个不同的2'羟基，其中用质子取代导致结合亲和力降低约10倍。相同的5个2'羟基降低了与几乎相同的水生栖热菌EF-Tu相互作用的亲和力。在tRNA（Phe）和T的共晶体结构中，观察到4个2'羟基形成氢键。aquaticus EF-Tu，而第五个2'羟基可以与tRNA中的分子内氢键缔合。然而，在晶体结构中观察到的与2'羟基的四个额外氢键在破坏时没有显示热力学效应。这些差异中的一些可以基于蛋白质和RNA的未结合结构来调和。(C)北京：科学出版社.
A set of 45 different tRNAs, each containing a single deoxynucleotide substitution covering the upper half of the molecule was used in conjunction with a high-throughput ribonuclease protection assay to investigate the thermodynamic role of 2' hydroxyl groups in stabilizing a complex with elongation factor Tu (EF-Tu) from Thermus thermophilus. Five distinct 2' hydroxyl groups were identified where substitution with a proton resulted in an approximately tenfold decrease in the binding affinity. The same five 2' hydroxyl groups reduced the affinity of the interaction with the nearly identical Thermus aquaticus EF-Tu. Four of these 2' hydroxyl groups were observed to form hydrogen bonds in a co-crystal structure of tRNA(Phe) and T. aquaticus EF-Tu, while the fifth 2' hydroxyl group can be associated with an intramolecular hydrogen bond in the tRNA. However, four additional hydrogen bonds to 2' hydroxyl groups observed in the crystal structure show no thermodynamic effect upon disruption. Some of these discrepancies may be reconciled based on the unbound structures of the protein and RNA. (C) 2001 Academic Press.