Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H.
Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H.
复制标题
HIV逆转录酶RNase H独特的渐进裂解机制。
DOI：
10.1073/pnas.210392297
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发表时间：
2000
影响因子：
11.1
通讯作者：
Bambara,RA
中科院分区：
文献类型：
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作者：
Wisniewski,M;Balakrishnan,M;Palaniappan,C;Fay,PJ;Bambara,RA
HIV-1逆转录酶（RT）降解正链病毒RNA基因组，同时合成负链DNA。许多RNA片段，包括多嘌呤片段，仍然与新的DNA退火。 据信，几种RT在合成后结合，以通过RNA酶H活性的聚合独立模式降解除多嘌呤段之外的所有RNA片段。对于后一个过程，我们发现RT将RNase H活性位点定位在距离RNA 5′端约18 nt的位置，进行初级切割。该酶重新结合或滑向RNA的5′端，进行二次切割，产生两个8-9 nt长的产物。然后RT结合由第一次或第二次切割产生的RNA的新5′端，以进行下一次主要切割。此外，我们观察到另一种类型的RNase H切割特异性。RT将RNase H活性位点与RNA的3′端对齐，切割5个残基。我们确定了这些削减的相对速率，定义了它们的时间顺序。结果表明，第一个主要的切割是最快的，和第二次和5-nt切割发生在类似的速度。 第二个主要切口出现在最后。基于这些结果，我们提出了一个模型，RT逐步切割RNA片段。
HIV-1 reverse transcriptase (RT) degrades the plus strand viral RNA genome while synthesizing the minus strand of DNA. Many RNA fragments, including the polypurine tracts, remain annealed to the new DNA. Several RTs are believed to bind after synthesis to degrade all RNA fragments except the polypurine tracts by a polymerization-independent mode of RNase H activity. For this latter process, we found that RT positions the RNase H active site approximately 18 nt from the 5′ end of the RNA, making the primary cut. The enzyme rebinds or slides toward the 5′ end of the RNA to make a secondary cut creating two products 8–9 nt long. RT then binds the new 5′ end of the RNA created by the first primary or the secondary cuts to make the next primary cut. In addition, we observed another type of RNase H cleavage specificity. RT aligns the RNase H active site to the 3′ end of the RNA, cutting 5 residues in. We determined the relative rates of these cuts, defining their temporal order. Results show that the first primary cut is fastest, and the secondary and 5-nt cuts occur next at similar rates. The second primary cuts appear last. Based on these results, we present a model by which RT progressively cleaves RNA fragments.