Physical mapping of HIV reverse transcriptase to the 5′ end of RNA primers
Physical mapping of HIV reverse transcriptase to the 5′ end of RNA primers
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DOI：
10.1074/jbc.m103958200
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发表时间：
2001-08-31
影响因子：
4.8
通讯作者：
Fitzgerald-Heath, MJ
中科院分区：
文献类型：
--
作者：
DeStefano, JJ;Cristofaro, JV;Fitzgerald-Heath, MJ
对RNA裂解产物的酶学分析表明，人类免疫缺陷病毒(HIV)逆转录酶(RT)与RNA的5‘端结合，而RNA的5’端嵌入较长的DNA模板(RNA引物)，但却与DNA引物的3‘端结合。一个令人担忧的问题是，结合在RNA 3‘端的RT分子不容易被检测到，因为当RT结合到3’端时，可能不会催化实质性的RNA延伸或切割。我们用物理作图的方法证明RT优先结合到RNA引物的5‘端。将缺乏RNase H活性的AM HIV-RT(HIV-RTE478Q)与RNA-DNA杂交体孵育，然后加入大肠杆菌RNase H，RT保护RNA 5‘端的23个碱基区和DNA链上的4个额外碱基。这种足迹与HIV-RT的晶体结构有很好的相关性。没有观察到RNA 3‘端的保护，尽管当包括dNTPs时，出现了低水平的延伸，表明RT可以结合这一末端。野生型HIV-RT切割了RNA，然后延伸了一小部分切割的片段，这表明非常小的RNA可能与DNA引物结合在一起。
Enzymatic analysis of RNA cleavage products has suggested that human immunodeficiency virus (HIV) reverse transcriptase (RT) binds to the 5' end of RNAs that are recessed on a longer DNA template (RNA primers) yet binds to the 3' end of DNA primers. One concern is that RT molecules bound at the 3' end of RNA would not be easily detected because RT may not catalyze substantial RNA extension or cleavage when bound to the 3' end. We used physical mapping to show that RT binds preferentially to the 5' end of RNA primers. Am HIV-RT that lacked RNase H activity (HIV-RTE478Q) was incubated with the RNA-DNA hybrid followed by the addition of Escherichia coli RNase H. RT protected a similar to 23-base region at the 5' end of the RNA and 4 additional bases on the DNA strand. This footprint correlated well with the crystal structure of HIV-RT. No protection of the RNA 3' end was observed, although when dNTPs were included, low levels of extension occurred, indicating that RT can bind this end. Wildtype HIV-RT cleaved the RNA and then extended a small portion of the cleaved fragments, suggesting that very small RNAs may be bound similar to DNA primers.