Job D, Job C, de Mercoyrol L, Shire D
Centre de Biochimie et de Biologie Moléculaire, Centre National de la Recherche Scientifique, Marseille, France.
Eur J Biochem. 1991 Feb 14;195(3):831-9. doi: 10.1111/j.1432-1033.1991.tb15772.x.
Interaction of purified eukaryotic RNA polymerase II with various synthetic palindromic DNA sequences is associated with the formation of transcriptional complexes of different stabilities, i.e. having different propensities for releasing the nascent transcript. This phenomenon was observed by using wheat-germ RNA polymerase II and a series of double-stranded template polymers containing palindromic repeating motifs of 6-16 bp, with regulatory alternating purine and pyrimidine bases such as d[ATA(CG)nC].d[TAT(GC)nG], with n = 1, 3 or 6 referred to as d(GC), d(GC)3 or d(GC)6, respectively. We also synthesized two double-stranded methylated polymers, containing the repeating units d(ATAm5CGm5C).d(TATGm5CG) and d[ATA(m5CG)6m5C].d[TAT(Gm5C)6G] [designated d(GmC) and d(GmC)6, respectively]. All of these polymers served as templates for the reaction of single-step addition of CTP to a CpG primer catalysed by wheat-germ RNA polymerase II, to an extent that seems well correlated with the number of potential initiation sites within the DNA molecules. Furthermore, in these reactions, the enzyme appears to form relatively stable transcriptional complexes, as trinucleotide product was released only very slowly. In marked contrast to the results with the CpG primer, the single-step addition reaction primed by UpA, i.e. the synthesis of UpApU proceeded at a much higher velocity and was strongly enhanced by increasing the d(G-C) content of the repeating units of the DNA polymers. Thus, taking into account the number of potential sites at which UpApU synthesis could occur, the extent of UpApU synthesis was increased about 12-fold with d(GC)6 compared to that with the d(GC) template. The catalytic nature of the reaction necessarily implies that the stability of the transcription complexes with the plant RNA polymerase II decreased as the d(G-C) content of the repeating motif increased. Furthermore, although the synthesis of CpGpC could be demonstrated with d(GmC)6 as template, the UpA-primed synthesis of UpApU could not be detected with this polymer. The results obtained in transcription of these polymers are discussed in relation to the potential involvement of palindromic DNA in transcription termination and attenuation in the presence of RNA polymerase II.
纯化的真核生物RNA聚合酶II与各种合成的回文DNA序列相互作用，与不同稳定性转录复合物的形成相关，即具有不同释放新生转录本的倾向。通过使用小麦胚芽RNA聚合酶II和一系列含有6 - 16个碱基对回文重复基序的双链模板聚合物观察到了这种现象，这些聚合物具有调节性的交替嘌呤和嘧啶碱基，如d[ATA(CG)nC].d[TAT(GC)nG]，其中n = 1、3或6，分别称为d(GC)、d(GC)3或d(GC)6。我们还合成了两种双链甲基化聚合物，分别含有重复单元d(ATAm5CGm5C).d(TATGm5CG)和d[ATA(m5CG)6m5C].d[TAT(Gm5C)6G] [分别命名为d(GmC)和d(GmC)6]。所有这些聚合物都作为模板，用于小麦胚芽RNA聚合酶II催化的将CTP单步添加到CpG引物的反应中，其程度似乎与DNA分子内潜在起始位点的数量密切相关。此外，在这些反应中，该酶似乎形成了相对稳定的转录复合物，因为三核苷酸产物释放得非常缓慢。与使用CpG引物的结果形成显著对比的是，由UpA引发的单步添加反应，即UpApU的合成速度要高得多，并且通过增加DNA聚合物重复单元的d(G - C)含量而得到强烈增强。因此，考虑到UpApU合成可能发生的潜在位点数量，与使用d(GC)模板相比，使用d(GC)6时UpApU的合成程度增加了约12倍。反应的催化性质必然意味着，随着重复基序的d(G - C)含量增加，与植物RNA聚合酶II形成的转录复合物的稳定性降低。此外，尽管可以证明以d(GmC)6为模板能合成CpGpC，但使用这种聚合物时无法检测到由UpA引发的UpApU的合成。结合RNA聚合酶II存在时回文DNA在转录终止和衰减中的潜在作用，讨论了这些聚合物转录所获得的结果。
