PHOTO-CROSS-LINKING OF THE SIGNAL SEQUENCE OF NASCENT PREPROLACTIN TO THE 54-KILODALTON POLYPEPTIDE OF THE SIGNAL RECOGNITION PARTICLE
PHOTO-CROSS-LINKING OF THE SIGNAL SEQUENCE OF NASCENT PREPROLACTIN TO THE 54-KILODALTON POLYPEPTIDE OF THE SIGNAL RECOGNITION PARTICLE
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DOI：
10.1073/pnas.83.22.8604
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发表时间：
1986-11-01
影响因子：
11.1
通讯作者：
JOHNSON, AE
中科院分区：
文献类型：
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作者：
KRIEG, UC;WALTER, P;JOHNSON, AE
在小麦胚芽蛋白合成系统中，光反应部分被加入到小麦胚芽蛋白合成系统中，方法是利用质粒源的前催乳素mRNA和Lys-tRNA类似物N.epsilon-(5-叠氮-2-硝基苯甲酰基)-Lys-tRNA。新生链中异常大的氨基酸侧链的存在不会损害功能：在没有信号识别颗粒(SRP)的情况下，完整的催乳素链被合成，在SRP的存在下，伸长被阻止，在盐提取的微体存在的情况下，观察到SRP依赖的跨内质网膜的易位和信号肽的切割。伸长受阻的核糖体的光解导致几个依赖于光和βANB-Lys-tRNA的交联物。通过使用针对每种蛋白质的抗体，一个共价复合体被证明是催乳素前幼发链和SRP的54 kDa蛋白亚基之间的光交联物。这表明，在伸长抑制的核糖体中，分泌蛋白的N端与SRP相邻，并强烈表明该信号序列可被SRP的54 kDa亚基识别并结合。其他的光交联涉及到大的核糖体亚单位中的未知蛋白质，这表明这种结合探针的方法提供了一种强有力的方法来研究环境以及内质网膜上的翻译和转位过程中新生链条的相互作用。Lys-tRNA类似物还成功地光亲和标记了EPB-Lys-tRNA.cntdot.EF-Tu.cntdot.GTP三元复合体中的大肠杆菌延长因子Tu(EF-Tu)。
Photoreactive moieties were incorporated into nascent polypeptides in a wheat germ protein-synthesizing system by using a plasmid-derived preprolactin mRNA and a Lys-tRNA analog, N.epsilon.-(5-azido-2-nitrobenzoyl)-Lys-tRNA (.epsilon.ANB-Lys-tRNA). The presence of the abnormally large amino acid side chains in the nascent chains did not impair function: complete preprolactin chains were synthesized in the absence of the signal recognition particle (SRP), elongation was arrested in the presence of SRP, and SRP-dependent translocation across the membrane of the endoplasmic reticulum and signal peptidase cleavage were observed in the presence of salt-extracted microsomes. Photolysis of elongation-arrested ribosomes resulted in several light- and .epsilon.ANB-Lys-tRNA-dependent crosslinks. By using antibodies specific for each of the proteins, one covalent complex was shown to be a photocrosslink between the preprolactin nascent chain and the 54-kDa protein subunit of SRP. This demonstrates that the N-terminal end of a secretory protein is located adjacent to the SRP in elongation-arrested ribosomes and strongly suggests that the signal sequence is recognized by and binds to the 54-kDa subunit of SRP. The other photocrosslinks involve as-yet-unidentified proteins in the large ribosomal subunit, indicating that this method of incorporating probes provides a powerful approach to examining the environment and interactions of the nascent chain during translation and translocation across the membrane of the endoplasmic reticulum. The Lys-tRNA analog also successfully photoaffinity-labeled the Escherichia coli enlongation factor Tu (EF-Tu) in the .epsilon.ANB-Lys-tRNA.cntdot.EF-Tu.cntdot.GTP ternary complex.