Bohjanen P R, Liu Y, Garcia-Blanco M A
Department of Pharmacology and Cancer Biology, Levine Science Research Center, Duke University Medical Center, Durham, NC 27710, USA.
Nucleic Acids Res. 1997 Nov 15;25(22):4481-6. doi: 10.1093/nar/25.22.4481.
The ability of the HIV-1 Tat protein to trans -activate HIV-1 transcription in vitro is specifically inhibited by a circular TAR RNA decoy. This inhibition is not overcome by adding an excess of Tat to the reaction but is partially overcome by adding Tat in combination with nuclear extract, suggesting that TAR RNA might function by interacting with a complex containing Tat and cellular factor(s). A cell-free transcription system involving immobilized DNA templates was used to further define the factor(s) that interact with TAR RNA. Preinitiation complexes formed in the presence or absence of Tat were purified on immobilized templates containing the HIV-1 promoter. After washing, nucleotides and radiolabelled UTP were added and transcription was measured. The presence of Tat during preinitiation complex formation resulted in an increase in the level of full-length HIV-1 transcripts. This Tat-activated increase in HIV-1 transcription was not inhibited by circular TAR decoys added during preinitiation complex formation but was inhibited by circular TAR decoys subsequently added during the transcription reaction. These results suggest that TAR decoys inhibit Tat-activated HIV-1 transcription after preinitiation complex formation, perhaps by interacting with components of transcription complexes.
HIV-1反式激活蛋白（Tat蛋白）在体外反式激活HIV-1转录的能力受到环状TAR RNA诱饵的特异性抑制。在反应中加入过量的Tat并不能克服这种抑制作用，但将Tat与核提取物联合加入时，这种抑制作用会部分被克服，这表明TAR RNA可能通过与包含Tat和细胞因子的复合物相互作用来发挥功能。利用一种涉及固定化DNA模板的无细胞转录系统进一步确定与TAR RNA相互作用的因子。在含有HIV-1启动子的固定化模板上纯化在有或无Tat存在的情况下形成的起始前复合物。洗涤后，加入核苷酸和放射性标记的UTP并测量转录情况。在起始前复合物形成过程中存在Tat会导致全长HIV-1转录本水平增加。在起始前复合物形成过程中加入环状TAR诱饵不会抑制这种由Tat激活的HIV-1转录增加，但在转录反应过程中随后加入环状TAR诱饵则会抑制这种增加。这些结果表明，TAR诱饵在起始前复合物形成后抑制由Tat激活的HIV-1转录，可能是通过与转录复合物的成分相互作用来实现的。
