Manche L, Green S R, Schmedt C, Mathews M B
Cold Spring Harbor Laboratory, New York 11724.
Mol Cell Biol. 1992 Nov;12(11):5238-48. doi: 10.1128/mcb.12.11.5238-5248.1992.
The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.
干扰素诱导蛋白激酶DAI，即双链RNA（dsRNA）激活的翻译抑制剂，在高等细胞中蛋白质合成的调控中起关键作用。一旦被激活，在一个涉及自身磷酸化的过程中，它会使起始因子eIF-2磷酸化，从而导致多肽链起始的抑制。DAI的活性受RNA调节剂控制，包括dsRNA激活剂和阻断dsRNA激活的高度结构化单链RNA。为了阐明激活机制，我们研究了DAI与不同大小的RNA双链体的相互作用。短于30bp的分子不能稳定结合且不能激活该酶，但在高浓度下它们会阻止长dsRNA的激活。长于30bp的分子能结合并激活该酶，其效率随链长增加而提高，在约85bp时达到最大值。这些dsRNA在高浓度下不能激活，并且也会阻止长dsRNA的激活。对dsRNA与DAI之间复合物的分析表明，在最大堆积时，该酶与低至单螺旋圈的dsRNA（11bp）相互作用，但在允许激活的条件下，结合位点可保护约80bp的双链体。当RNA结合位点被RNA激活剂完全占据时，复合物似乎会发生构象变化。
