Pata Janice D, King Bradford R, Steitz Thomas A
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Nucleic Acids Res. 2002 Nov 15;30(22):4855-63. doi: 10.1093/nar/gkf620.
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates DNA synthesis from the 3' end of human tRNA(Lys3). We have used cis-acting hammerhead ribozymes to produce homogeneous-length transcribed tRNA(Lys3) and 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 tRNA(Lys3), 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 vitro transcribed 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 this in vitro transcribed tRNA(Lys3) to grow.
1型人类免疫缺陷病毒（HIV-1）逆转录酶（RT）从人tRNA（Lys3）的3'末端起始DNA合成。我们利用顺式作用锤头状核酶产生均一长度的转录tRNA（Lys3），并开发了在改良的管凝胶装置上纯化高度结构化RNA的条件。滴定实验表明，这种RNA能组装成一个起始复合物，该复合物包含等摩尔量的HIV-1 RT、转录的tRNA（Lys3）和化学合成的模板RNA。我们使用凝胶过滤色谱法纯化了该复合物，发现其分子量均一，这表明起始复合物在微摩尔浓度下形成单一离散物种。当向该起始复合物提供脱氧核苷酸时，基本上所有的tRNA都被HIV-1 RT用作引物，并完全延伸至模板的5'末端。因此，体外转录的tRNA可被HIV-1 RT有效地用作引物。我们还获得了HIV-1起始复合物的晶体，这些晶体需要这种体外转录的tRNA（Lys3）的精确界定末端才能生长。
