Studies on three E-coli DEAD-box helicases point to an unwinding mechanism different from that of model DNA helicases
Studies on three E-coli DEAD-box helicases point to an unwinding mechanism different from that of model DNA helicases
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DOI：
10.1021/bi049852s
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发表时间：
2004-06-22
期刊：
影响因子：
2.9
通讯作者：
Dreyfus, M
中科院分区：
文献类型：
--
作者：
Bizebard, T;Ferlenghi, I;Dreyfus, M
死亡盒蛋白参与所有生物体RNA代谢的各个方面。这些RNA依赖性ATP酶通常被认为是双链RNA解旋酶，尽管在体外这种活性仅被证明是其中的一个子集。鉴于其高度的生物特异性，其可疑的解旋活性可能反映了体外使用的底物的非同源特性。在这里，我们指出这种难以捉摸的其他原因。比较了三种E. coliDEAD-box蛋白、CsdA、RhlE和SrmB。尽管所有蛋白质的ATP酶活性都被长RNA刺激（尽管程度不同），但只有RhlE被短寡核糖核苷酸刺激。一致地，所有三种蛋白质都可以解开具有长单链延伸的RNA双链体，但是当延伸短或不存在时，只有RhlE是有效的。另一个关键的限制因素涉及双链体区域的长度：在RhlE的情况下，比率（双链体解绕）/（ATP水解）从11个碱基对下降到14个碱基对，下降1000倍，表明低持续合成能力。值得注意的是，考虑到这些限制，所有三种蛋白质都可以用5'或3'延伸（或在RhlE的情况下没有延伸）解开底物。这种行为，这与充分研究的SFI DNA解旋酶，进行了讨论，在现有的结构和生化数据。
DEAD-box proteins participate in various aspects of RNA metabolism in all organisms. These RNA-dependent ATPases are usually regarded as double-stranded RNA unwinding enzymes, though in vitro this activity has only been demonstrated for a subset of them. Given their high biological specificity, their equivocal unwinding activity may reflect the noncognate character of the substrates used in vitro. Here, we pinpoint other reasons for this elusiveness. We have compared the ATPase and helicase activities of three E. coli DEAD-box proteins, CsdA, RhlE and SrmB. Whereas the ATPase activity of all proteins is stimulated (albeit to various degree) by long RNAs, only RhlE is stimulated by short oligoribonucleotides. Consistently, all three proteins can unwind RNA duplexes with long single-stranded extensions, but only RhlE is effective when extensions are short or absent. Another critical constraint concerns the length of the duplex region: in the case of RhlE, the ratio (duplex unwound)/(ATP hydrolyzed) drops 1000-fold upon going, from 11 to 14 base pairs, indicating a low processivity. Remarkably, allowing for these constraints, all three proteins can unwind substrates with either 5' or 3' extensions (or no extension in the case of RhlE). This behavior, which contrasts with that of well studied SFI DNA helicases, is discussed in the light of available structural and biochemical data.