Zakharova O D, Tarrago-Litvak L, Fournier M, Litvak S, Nevinsky G A
Institute of Bioorganic Chemistry, Siberian Division of the Academy of Sciences of Russia, Novosibirsk, Russian Federation.
FEBS Lett. 1995 Oct 16;373(3):255-8. doi: 10.1016/0014-5793(95)01056-k.
HIV-1 RT is able to catalyze DNA synthesis starting from mononucleotides used both as minimal primers and as nucleotide substrates (de novo synthesis) in the presence of a complementary template. The rate of this process is rather slow when compared to the polymerization primed by an oligonucleotide. The addition of tRNA(Lys,3) to this system increased the de novo synthesis rate by 2-fold. Addition of low concentrations of agents able to modify protein conformation, such as urea, dimethylsulfoxide and Triton X-100, can activate the de novo synthesis by a factor 2 to 5. A dramatic synergy is observed in the presence of the three compounds since the stimulating effect of tRNA increases 10-15 times. These results suggest that compounds activating RT are able to induce a conformational change of the enzyme which results in a higher specific activity. Primer tRNA seems to play an important role in HIV-1 RT modification(s) leading to a polymerase having a higher affinity for the primer or the dTTP, but not for the template. The specificity of RT for the template is not influenced by changes in the kinetics or in the thermodynamic parameters of the polymerization reaction.
HIV-1逆转录酶能够在存在互补模板的情况下，从既用作最小引物又用作核苷酸底物的单核苷酸开始催化DNA合成（从头合成）。与由寡核苷酸引发的聚合反应相比，这个过程的速率相当缓慢。向该系统中添加tRNA（Lys，3）可使从头合成速率提高2倍。添加低浓度的能够改变蛋白质构象的试剂，如尿素、二甲基亚砜和吐温X-100，可使从头合成活性提高2至5倍。在存在这三种化合物的情况下观察到显著的协同作用，因为tRNA的刺激作用增加了10至15倍。这些结果表明，激活逆转录酶的化合物能够诱导酶的构象变化，从而导致更高的比活性。引物tRNA似乎在HIV-1逆转录酶的修饰中起重要作用，这种修饰导致聚合酶对引物或dTTP具有更高的亲和力，但对模板没有更高的亲和力。逆转录酶对模板的特异性不受聚合反应动力学或热力学参数变化的影响。
