Assembly, purification and crystallization of an active HIV-1 reverse transcriptase initiation complex.
Assembly, purification and crystallization of an active HIV-1 reverse transcriptase initiation complex.
复制标题
活性 HIV-1 逆转录酶起始复合物的组装、纯化和结晶。
DOI：
10.1093/nar/gkf620
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发表时间：
2002
影响因子：
14.9
通讯作者：
Steitz,ThomasA
中科院分区：
文献类型：
--
作者：
Pata,JaniceD;King,BradfordR;Steitz,ThomasA
Human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) initiates DNA synthesis from the 3′ end of human tRNALys3. We have usedcis‐acting hammerhead ribozymes to produce homogeneous‐length transcribed tRNALys3and have developed conditions for purifying highly structured RNAs on a modified tube‐gel apparatus. Titration experiments show that this RNA can assemble into an initiation complex that contains equimolar amounts of HIV‐1 RT, transcribed tRNALys3, and chemically synthesized template RNA. We have purified this complex using gel‐filtration chromatography and have found that it is homogeneous with respect to molecular weight, demonstrating that the initiation complex forms a single discrete species at micromolar concentrations. When this initiation complex is supplied with deoxynucleotides, essentially all of the tRNA is used as a primer by HIV‐1 RT and is fully extended to the 5′ end of the template. Thus,in vitrotranscribed tRNA can be used efficiently as a primer by HIV‐1 RT. We have also obtained crystals of the HIV‐1 initiation complex that require the precisely defined ends of thisin vitrotranscribed tRNALys3to grow.
