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首页> 外文期刊>Molecular and Cellular Biology >The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis.
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The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis.

机译：DEAD盒RNA解旋酶真核翻译起始因子4A的HRIGRXXR区是RNA结合和ATP水解所必需的。

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eIF-4A is a eukaryotic translation initiation factor that is required for mRNA binding to ribosomes. It exhibits single-stranded RNA-dependent ATPase activity, and in combination with a second initiation factor, eIF-4B, it exhibits duplex RNA helicase activity. eIF-4A is the prototype of a large family of proteins termed the DEAD box protein family, whose members share nine highly conserved amino acid regions. The functions of several of these conserved regions in eIF-4A have previously been assigned to ATP binding, ATPase, and helicase activities. To define the RNA-binding region of eIF-4A, a UV-induced cross-linking assay was used to analyze binding of mutant eIF-4A proteins to RNA. Mutants carrying mutations in the ATP-binding region (AXXXXGKT), ATPase region (DEAD), helicase region (SAT), and the most carboxy-terminal conserved region of the DEAD family, HRIGRXXR, were tested for RNA cross-linking. We show that mutations, either conservative or not, in any one of the three arginines in the HRIGRXXR sequence drastically reduced eIF-4A cross-linking to RNA. In addition, all the mutations in the HRIGRXXR region abrogate RNA helicase activity. Some but not all of these mutations affect ATP binding and ATPase activity. This is consistent with the hypothesis that the HRIGRXXR region is involved in the ATP hydrolysis reaction and would explain the coupling of ATPase and RNA-binding/helicase activities. Our results show that the HRIGRXXR region, which is QRXGRXXR or QXXGRXXR in the RNA and DNA helicases of the helicase superfamily II, is involved in ATP hydrolysis-dependent RNA interaction during unwinding. We also show that mutations in other regions of eIF-4A that abolish ATPase activity sharply decrease eIF-4A cross-linking to RNA. A model is proposed in which eIF-4A first binds ATP, resulting in a change in eIF-4A conformation which allows RNA binding that is dependent on the HRIGRXXR region. Binding of RNA induces ATP hydrolysis, leading to a more stable interaction with RNA. This process is then linked to unwinding of duplex RNA in the presence of eIF-4B.
机译：eIF-4A是真核翻译起始因子，是mRNA与核糖体结合所必需的。它具有单链RNA依赖性ATPase活性，并且与第二个起始因子eIF-4B结合，具有双链RNA解旋酶活性。 eIF-4A是称为DEAD盒蛋白家族的一大类蛋白的原型，其成员共有9个高度保守的氨基酸区域。先前已将eIF-4A中这些保守区中的几个保守区的功能分配给ATP结合，ATPase和解旋酶活性。为了定义eIF-4A的RNA结合区域，使用UV诱导的交联测定法分析突变的eIF-4A蛋白与RNA的结合。测试了在ATP结合区域（AXXXXGKT），ATPase区域（DEAD），解旋酶区域（SAT）和DEAD家族最羧基末端保守的区域HRIGRXXR中携带突变的突变体的RNA交联。我们显示，在HRIGRXXR序列的三个精氨酸中的任何一个中，无论保守与否，突变都显着降低了eIF-4A与RNA的交联。另外，HRIGRXXR区的所有突变都消除了RNA解旋酶的活性。这些突变中的一些但不是全部都会影响ATP结合和ATPase活性。这与HRIGRXXR区域参与ATP水解反应的假设相符，并且可以解释ATPase和RNA结合/解旋酶活性的耦合。我们的结果表明，解旋酶超家族II的RNA和DNA解旋酶中的HRIGRXXR区为QRXGRXXR或QXXGRXXR，在放卷过程中参与了ATP水解依赖性RNA相互作用。我们还显示，在废除ATPase活性的eIF-4A其他区域中的突变会急剧降低eIF-4A与RNA的交联。提出了一种模型，其中eIF-4A首先结合ATP，导致eIF-4A构象发生变化，从而允许依赖HRIGRXXR区域的RNA结合。 RNA的结合诱导ATP水解，从而导致与RNA的相互作用更加稳定。然后，此过程与存在eIF-4B时解链双链体RNA有关。

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