Insights into how RNase R degrades structured RNA: analysis of the nuclease domain.
Insights into how RNase R degrades structured RNA: analysis of the nuclease domain.
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DOI：
10.1016/j.jmb.2009.01.068
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发表时间：
2009-04-03
影响因子：
5.6
通讯作者：
Deutscher, Murray P.
中科院分区：
文献类型：
--
作者：
Vincent, Helen A.;Deutscher, Murray P.
RNase R很容易降解高度结构化的RNA，而它的同源物RNase II却不能这样做。此外，RNase R的核酸酶结构域缺乏所有典型的RNA结合结构域，足以实现这种活性。RNase R也比RNase II在其催化通道内更紧密地结合RNA，这被认为是其独特催化特性的重要原因。为了进一步研究这一想法，RNase R的核酸酶结构域通道内的某些残基被改变为RNase II中的残基。在众多研究中，我们发现了一种氨基酸R572，它在RNase r的特性中起着重要作用。在RNase II中发现，将这种残基转化为赖氨酸，导致在核酸酶结构域通道内较弱的底物结合，较长的极限产物，对各种底物的活性增加，以及更快的底物吸收速率。最重要的是，突变体在降解结构RNA时遇到困难，在双链区域内暂停。其他研究表明，结构底物的降解依赖于温度，这表明热呼吸在RNase R的作用机制中起作用。基于这些数据，我们提出了一个模型，其中核酸酶结构域内的紧密结合允许RNase R利用RNA双工的自然热呼吸来降解结构RNA。
RNase R readily degrades highly structured RNA, whereas its paralogue, RNase II, is unable to do so. Furthermore, the nuclease domain of RNase R, devoid of all canonical RNA binding domains, is sufficient for this activity. RNase R also binds RNA more tightly within its catalytic channel than does RNase II, which is thought to be important for its unique catalytic properties. To investigate this idea further, certain residues within the nuclease domain channel of RNase R were changed to those found in RNase II. Among the many examined, we identified one amino acid, R572, that plays a significant role in the properties of RNase R. Conversion of this residue to lysine, as found in RNase II, results in weaker substrate binding within the nuclease domain channel, longer limit products, increased activity against a variety of substrates and a faster substrate on-rate. Most importantly, the mutant encounters difficulty in degrading structured RNA, pausing within a double-stranded region. Additional studies show that degradation of structured substrates is dependent upon temperature, suggesting a role for thermal breathing in the mechanism of action of RNase R. Based on these data, we propose a model in which tight binding within the nuclease domain allows RNase R to capitalize on the natural thermal breathing of an RNA duplex to degrade structured RNAs.
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