Komar A A, Kommer A, Krasheninnikov I A, Spirin A S
Department of Molecular Biology, Faculty of Biology, Moscow State University, 119899 Moscow, Russia.
J Biol Chem. 1997 Apr 18;272(16):10646-51. doi: 10.1074/jbc.272.16.10646.
Globin synthesis in a wheat germ cell-free translation system was performed in the presence of [3H]hemin and [35S]methionine to determine the minimal length of the nascent ribosome-bound globin chain capable of heme binding. Nascent polypeptides of predetermined size were synthesized on ribosomes by translation of truncated mRNA molecules. Analysis with the use of sucrose gradient centrifugation and puromycin reaction revealed that the ribosome-bound N-terminal alpha-globin fragments of 140, 100, and 86 amino acid residues are capable of an efficient heme binding, whereas those of 75, 65, and 34 amino acid residues display a significantly weaker, or just nonspecific, affinity to heme. This indicates that the ribosome-bound nascent chain of 86 amino acid residues has already acquired a spatial structure that allows its interaction with the heme group or that heme attachment promotes the formation of the proper tertiary structure in the ribosome-bound nascent peptide. In any case the cotranslational folding of globin is suggested.
在[3H]血红素和[35S]甲硫氨酸存在的情况下，在小麦胚芽无细胞翻译系统中进行珠蛋白合成，以确定能够结合血红素的新生核糖体结合珠蛋白链的最小长度。通过截短的mRNA分子的翻译在核糖体上合成预定大小的新生多肽。使用蔗糖梯度离心和嘌呤霉素反应进行分析表明，具有140、100和86个氨基酸残基的核糖体结合的N端α-珠蛋白片段能够有效结合血红素，而具有75、65和34个氨基酸残基的片段对血红素的亲和力明显较弱或仅表现为非特异性。这表明具有86个氨基酸残基的核糖体结合新生链已经获得了允许其与血红素基团相互作用的空间结构，或者血红素附着促进了核糖体结合新生肽中正确三级结构的形成。无论如何，提示珠蛋白存在共翻译折叠。
