Hesterkamp T, Hauser S, Lütcke H, Bukau B
Zentrum für Molekulare Biologie, Universität Heidelberg, Germany.
Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4437-41. doi: 10.1073/pnas.93.9.4437.
Correct folding of newly synthesized proteins is proposed to be assisted by molecular chaperones and folding catalysts. To identify cellular factors involved in the initial stages of this process we searched for proteins associated with nascent polypeptide chains. In an Escherichia coli transcription/translation system synthesizing beta-galactosidase we identified a 58-kDa protein which associated with translating ribosomes but dissociated from these ribosomes upon release of nascent beta-galactosidase. N-terminal sequencing identified it as trigger factor, previously implicated in protein secretion. Direct evidence for association of trigger factor with nascent polypeptide chains was obtained by crosslinking. In a wheat germ translation system complemented with E. coli lysates, epsilon-4-(3-trifluoromethyldiazirino)benzoic acid-lysine residues were incorporated into nascent secretory preprolactin and a nonsecretory preprolactin mutant. Trigger factor crosslinked to both types of nascent chains, provided they were ribosome bound. Trigger factor contains key residues of the substrate-binding pocket of FK506-binding protein-type peptidyl-prolyl-cis/trans-isomerases and has prolyl isomerase activity in vitro. We propose that trigger factor is a folding catalyst acting cotranslationally.
新合成蛋白质的正确折叠被认为是由分子伴侣和折叠催化剂协助完成的。为了鉴定参与这一过程初始阶段的细胞因子，我们寻找了与新生多肽链相关的蛋白质。在一个合成β-半乳糖苷酶的大肠杆菌转录/翻译系统中，我们鉴定出一种58 kDa的蛋白质，它与正在翻译的核糖体结合，但在新生β-半乳糖苷酶释放后从这些核糖体上解离。N端测序确定它为触发因子，先前认为它与蛋白质分泌有关。通过交联获得了触发因子与新生多肽链相关的直接证据。在补充了大肠杆菌裂解物的麦胚翻译系统中，将ε-4-(3-三氟甲基重氮苯甲酰基)苯甲酸-赖氨酸残基掺入新生的分泌型前催乳素和非分泌型前催乳素突变体中。只要新生链与核糖体结合，触发因子就会与这两种类型的新生链交联。触发因子含有FK506结合蛋白型肽基脯氨酰顺/反异构酶底物结合口袋的关键残基，并且在体外具有脯氨酰异构酶活性。我们认为触发因子是一种在共翻译过程中起作用的折叠催化剂。
