Schwartz R J, Tsai S Y, O'Malley B W
J Biol Chem. 1975 Jul 10;250(13):5165-74.
The interaction of Escherichia coli RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6.) with chick oviduct DNA and chromatin has been studied in order to quantify the RNA polymerase binding sites and rifampicin-resistant initiation sites. The number of high affinity binding sites at saturation was calculated from the number of molecules of enzyme bound to DNA or to chromatin. The number of rifampicin-resistant initiation sites was calculated from the total quantity and the number average chain length of RNA synthesized under the conditions in which only RNA polymerase already in a stable preinitiation complex could avoid inhibition by the drug. Using an assessment of the temperature requirement for the information of this preinitiation complex, we were able to differentiate initiations at single-stranded or nicked DNA regions from initiations at double-stranded DNA regions. This method enabled us to rule out the possibility that RNA chain initiation on chromatin was due to initiation at nicked or end regions of DNA. In addition, we also demonstrated that not all RNA polymerase molecules bound to DNA or chromatin are located at a site at which RNA chain initiation can immediately begin. This methodological approach should allow investigators to monitor the individual reactions and factors involved in in vitro transcription of eukaryotic DNA and chromatin.
为了量化RNA聚合酶结合位点和利福平抗性起始位点，对大肠杆菌RNA聚合酶（核糖核苷三磷酸：RNA核苷酸转移酶，EC 2.7.7.6.）与鸡输卵管DNA和染色质的相互作用进行了研究。根据与DNA或染色质结合的酶分子数量计算饱和时高亲和力结合位点的数量。利福平抗性起始位点的数量是根据在仅已处于稳定起始前复合物中的RNA聚合酶能够避免药物抑制的条件下合成的RNA的总量和数均链长计算得出的。通过评估形成该起始前复合物所需的温度条件，我们能够区分在单链或切口DNA区域的起始与在双链DNA区域的起始。该方法使我们能够排除染色质上RNA链起始是由于DNA切口或末端区域起始的可能性。此外，我们还证明并非所有与DNA或染色质结合的RNA聚合酶分子都位于RNA链起始可立即开始的位点。这种方法应该能让研究人员监测真核DNA和染色质体外转录中涉及的各个反应和因素。
