Evaluation of the RNA determinants for bacterial and yeast RNase III binding and cleavage
Evaluation of the RNA determinants for bacterial and yeast RNase III binding and cleavage
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DOI：
10.1074/jbc.m309324200
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发表时间：
2004-01-16
影响因子：
4.8
通讯作者：
Abou Elela, S
中科院分区：
文献类型：
--
作者：
Lamontagne, B;Abou Elela, S
细菌双链RNA特异性RNase III识别RNA螺旋的A型，几乎没有序列特异性。相比之下，面包酵母RNase III（Rnt1p）选择性地识别NGNN四环，即使它们连接到B型DNA螺旋。为了理解RNase III底物识别的一般机制，我们绘制了Rnt1p结合信号，并直接将其底物特异性与大肠杆菌RNase III和裂殖酵母RNase III（PacI）进行了比较。Rnt1p结合，但不切割长RNA双链体没有NGNN四环，而RNase III不加区别地切割所有的RNA双链体。在生理条件下，PacI切割RNA双链体，对NGNN加帽的RNA茎有一定的偏好。羟基自由基足迹表明Rnt1p特异性地与NGNN四环及其周围的核苷酸相互作用。相反，Rnt1p与GAAA帽发夹的相互作用很弱，很大程度上是非特异性的。PacI具有一定的底物识别双重性，但细菌RNase III没有。e. coli RNase III识别长度超过11 bp的RNA双链体，但特异性很低，切割不需要特异性特征。另一方面，PacI切割长的，但不是短的，RNA双链体，几乎没有序列特异性。短于27 bp的RNA茎的PacI切割依赖于切割位点上游两个核苷酸的UU-UC内环的存在。这些观察结果表明，酵母RNase III有两种识别机制，一种是使用特定的结构特征，另一种是识别A型RNA螺旋的一般特征。
Bacterial double-stranded RNA-specific RNase III recognizes the A-form of an RNA helix with little sequence specificity. In contrast, baker yeast RNase III (Rnt1p) selectively recognizes NGNN tetraloops even when they are attached to a B-form DNA helix. To comprehend the general mechanism of RNase III substrate recognition, we mapped the Rnt1p binding signal and directly compared its substrate specificity to that of both Escherichia coli RNase III and fission yeast RNase III (PacI). Rnt1p bound but did not cleave long RNA duplexes without NGNN tetraloops, whereas RNase III indiscriminately cleaved all RNA duplexes. PacI cleaved RNA duplexes with some preferences for NGNN-capped RNA stems under physiological conditions. Hydroxyl radical footprints indicate that Rnt1p specifically interacts with the NGNN tetraloop and its surrounding nucleotides. In contrast, Rnt1p interaction with GAAA-capped hairpins was weak and largely unspecific. Certain duality of substrate recognition was exhibited by PacI but not by bacterial RNase III. E. coli RNase III recognized RNA duplexes longer than 11 bp with little specificity, and no specific features were required for cleavage. On the other hand, PacI cleaved long, but not short, RNA duplexes with little sequence specificity. PacI cleavage of RNA stems shorter than 27 bp was dependent on the presence of an UU-UC internal loop two nucleotides upstream of the cleavage site. These observations suggest that yeast RNase IIIs have two recognition mechanisms, one that uses specific structural features and another that recognizes general features of the A-form RNA helix.