Photoaffinity polyamines:: interactions with AcPhe-tRNA free in solution or bound at the P-site of Escherichia coli ribosomes
Photoaffinity polyamines:: interactions with AcPhe-tRNA free in solution or bound at the P-site of Escherichia coli ribosomes
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DOI：
10.1093/nar/28.19.3733
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发表时间：
2000-10-01
影响因子：
14.9
通讯作者：
Kalpaxis, DL
中科院分区：
文献类型：
--
作者：
Amarantos, I;Kalpaxis, DL
精胺的两种光反应衍生物，叠氮基苯甲脒 (ABA)-精胺和硝基苯甲酰 (ANB)-精胺，用于绘制溶液中游离的 AcPhe-tRNA 或结合在大肠杆菌聚 (U) 编程核糖体 P 位点上的多胺结合位点。部分核酸酶消化表明，由 D 茎和可变环的核苷形成的深袋以及反密码子茎是游离状态下 AcPhe-tRNA 的优选多胺结合位点。 ABA-精胺是比ANB-精胺更强的交联剂。当 AcPhe-tRNA 非酶促结合到聚（U）编程核糖体时，两种光探针都以更高的亲和力连接，特别是，T psi C 茎和受体茎处的交联显着促进，光标记的 AcPhe-tRNA.聚（U）核糖体复合物对嘌呤霉素表现出中等反应性，光探针与 AcPhe-tRNA 的附着是造成这种缺陷的主要原因。当AcPhe-tRNA聚(U)核糖体复合物在翻译因子存在的情况下形成时，揭示了更复杂的情况；在50μM光探针存在下，通过照射这种复合物，刺激了对嘌呤霉素的反应性，较高浓度则具有抑制作用，刺激作用与光探针与核糖体的结合密切相关，结果是在光探针掺入可能引起的AcPhe-tRNA构象变化的基础上进行讨论的。
Two photoreactive derivatives of spermine, azido-benzamidino (ABA)-spermine and atidonitrobenzoyl (ANB)-spermine, were used for mapping of polyamine binding sites in AcPhe-tRNA free in solution or bound at the P-site of Escherichia coli poly(U)programmed ribosomes. Partial nuclease digestion indicated that the deep pocket formed by nucleosides of the D-stem and the variable loop, as well as the anticodon stem, are preferable polyamine binding sites for AcPhe-tRNA in the free state. ABA-spermine was a stronger cross-linker than ANB-spermine. Both photoprobes were linked to AcPhe-tRNA with higher affinity when the latter was nonenzymatically bound to poly(U)-programmed ribosomes, In particular, the cross-linking at the T psi C stem and acceptor stem was substantially promoted, The photolabeled AcPhe-tRNA.poly(U)ribosome complex exhibited moderate reactivity towards puromycin, The attachment of photoprobes to AcPhe-tRNA was mainly responsible for this defect. A more complicated situation was revealed when the AcPhe-tRNA poly(U) ribosome complex was formed in the presence of translation factors; the reactivity towards puromycin was stimulated by irradiating such a complex in the presence of photoprobes at 50 mu M, with higher concentrations being inhibitory, The stimulatory effect was closely related with the binding of photoprobes to ribosomes, The results are discussed on the basis of possible AcPhe-tRNA conformational changes induced by the incorporation of photoprobes.