THE HRIGRXXR REGION OF THE DEAD BOX RNA HELICASE EUKARYOTIC TRANSLATION INITIATION FACTOR-4A IS REQUIRED FOR RNA-BINDING AND ATP HYDROLYSIS
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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DOI：
10.1128/mcb.13.11.6789
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发表时间：
1993-11-01
影响因子：
5.3
通讯作者：
SONENBERG, N
中科院分区：
文献类型：
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作者：
PAUSE, A;METHOT, N;SONENBERG, N
eIF-4A是一种真核生物翻译起始因子，是mRNA与核糖体结合所必需的。它表现出单链RNA依赖性ATP酶活性，并与第二个起始因子eIF-4 B结合，表现出双链体RNA解旋酶活性。eIF-4A是称为DEAD盒蛋白家族的蛋白质大家族的原型，其成员共享9个高度保守的氨基酸区域。eIF-4A中这些保守区域中的几个的功能先前已被分配给ATP结合、ATP酶和解旋酶活性。为了确定eIF-4A的RNA结合区域，使用UV诱导的交联测定来分析突变体eIF-4A蛋白与RNA的结合。对在ATP结合区（AXXXXGKT）、ATP酶区（DEAD）、解旋酶区（SAT）和DEAD家族的最羧基末端保守区HRIGRXXR中携带突变的突变体进行RNA交联测试。我们发现，在HRIGRXXR序列中的三个碱基中的任何一个中的突变，无论是保守的还是不保守的，都会大大减少eIF-4A与RNA的交联。此外，HRIGRXXR区域中的所有突变消除RNA解旋酶活性。这些突变中的一些但不是全部影响ATP结合和ATP酶活性。这与HRIGRXXR区域参与ATP水解反应的假设一致，并将解释ATP酶和RNA结合/解旋酶活性的偶联。我们的研究结果表明，HRIGRXXR区域，这是QRXGRXXR或QXXGRXXR在RNA和DNA解旋酶的解旋酶超家族II，是参与ATP水解依赖的RNA相互作用在解旋。我们还发现，eIF-4A的其他区域的突变，废除ATP酶活性急剧减少eIF-4A与RNA的交联。提出了一个模型，其中eIF-4A首先结合ATP，导致eIF-4A构象的变化，这使得RNA结合依赖于HRIGRXXR区域。RNA的结合诱导ATP水解，导致与RNA更稳定的相互作用。然后，该过程与在eIF-4 B存在下双链体RNA的解旋相关联。
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 reduce 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.