Phillips D R, Crothers D M
Biochemistry. 1986 Nov 18;25(23):7355-62. doi: 10.1021/bi00371a017.
An assay has been developed to detect the DNA sequence specificity of drug binding sites, and the kinetics of dissociation of drug from those sites, under conditions involving active transcription of the DNA. Specific transcriptional blockage sites were detected in the presence of actinomycin D and a bisintercalator, bis(anthracycline); the rate of RNA chain growth past the drug binding sites yields the rate of dissociation of drug from these sites. Rate constants for dissociation from the whole promoter fragment measured by the detergent sequestration method were found to be significantly faster than the rate determined for dissociation from the specific transcriptional blockage site. However, the absence of significant blockage at other drug binding sites implies much more rapid drug dissociation from those sites in the transcriptional complex. We conclude that transcriptional blockage results from a DNA sequence-dependent interaction of the drug-DNA complex with RNA polymerase; the sequences that are effective for blockage appear to be GpC for actinomycin and (CpA)3 for a bis(daunomycin) compound. Transcriptional inhibition may in general show greater sequence specificity than is exhibited by simple binding.
已经开发出一种检测方法，用于在涉及DNA活跃转录的条件下，检测药物结合位点的DNA序列特异性以及药物从这些位点解离的动力学。在放线菌素D和双嵌入剂双（蒽环类）存在的情况下，检测到了特定的转录阻断位点；RNA链越过药物结合位点的生长速率产生了药物从这些位点的解离速率。通过去污剂隔离法测量的从整个启动子片段解离的速率常数，发现明显快于从特定转录阻断位点解离所确定的速率。然而，在其他药物结合位点没有明显的阻断，这意味着药物在转录复合物中从这些位点的解离要快得多。我们得出结论，转录阻断是由药物-DNA复合物与RNA聚合酶的DNA序列依赖性相互作用引起的；对放线菌素有效的阻断序列似乎是GpC，对双（柔红霉素）化合物是（CpA）3。一般来说，转录抑制可能比简单结合表现出更大的序列特异性。
