Landgraf R, Ramamurthi K S, Sigman D S
Department of Chemistry and Biochemistry, Molecular Biology Institute, University of California, Los Angeles 90024-1570, USA.
Nucleic Acids Res. 1996 Aug 15;24(16):3246-52. doi: 10.1093/nar/24.16.3246.
We have used R-loop formation and direct hybridization techniques to analyze the kinetics by which RNA is displaced from a heteroduplex by DNA of identical sequence. Using random walk simulations we were able to calculate the step times for a single displacement reaction. For RNA with a GC content of 57-60% the data indicate an RNA exchange probability of 50.06%, which is indicative of a modest destabilization of the heteroduplex compared with a DNA duplex in the presence of magnesium. The average step time for the reversible exchange of a single nucleotide is 345.0 (+/- 1.3) ms/step. An acceleration of the displacement reaction was observed in the absence of magnesium. A comparison with step times for elongation shows that RNA displacement would not be rate limiting to transcription elongation under two conditions: (i) if magnesium is eliminated from the newly synthesized heteroduplex; (ii) if displacement is kept in a forward only exchange mode through binding of the emerging RNA. Distamycin, a minor groove binding drug, is very effective as a 'catalyst' of RNA displacement. This effect is likely to be due to preferential binding of distamycin to the minor groove of the DNA duplex as opposed to the heteroduplex. This kinetic assay could therefore serve as a convenient assay for the determination of binding preferences of nucleic acid ligands.
我们使用R环形成和直接杂交技术来分析相同序列的DNA将RNA从异源双链体中置换出来的动力学过程。通过随机游走模拟，我们能够计算单个置换反应的步长时间。对于GC含量为57 - 60%的RNA，数据表明RNA交换概率为50.06%，这表明与存在镁时的DNA双链体相比，异源双链体的稳定性略有降低。单个核苷酸可逆交换的平均步长时间为345.0（±1.3）毫秒/步。在没有镁的情况下观察到置换反应加速。与延伸步长时间的比较表明，在两种情况下RNA置换不会成为转录延伸的限速因素：（i）如果从新合成的异源双链体中去除镁；（ii）如果通过新出现的RNA的结合使置换保持仅向前交换模式。小沟结合药物Distamycin作为RNA置换的“催化剂”非常有效。这种效应可能是由于Distamycin优先结合DNA双链体的小沟而非异源双链体。因此，这种动力学测定可作为一种方便的测定方法，用于确定核酸配体的结合偏好。
