RNA polymerase alters the mobility of an A-residue crucial to polymerase-induced melting of promoter DNA.
RNA polymerase alters the mobility of an A-residue crucial to polymerase-induced melting of promoter DNA.
复制标题
RNA 聚合酶改变 A 残基的迁移率，这对聚合酶诱导的启动子 DNA 解链至关重要。
DOI：
10.1021/bi026539m
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
DeHaseth,PieterL
中科院分区：
文献类型：
--
作者：
Tsujikawa,Laura;Strainic,MichaelG;Watrob,Heather;Barkley,MaryD;DeHaseth,PieterL
大肠杆菌RNA聚合酶诱导的启动子DNA链分离可能起始于非模板链-11位保守的A残基。在这里，我们描述了使用荧光技术来研究RNA聚合酶与-11碱基的相互作用。使用叉状DNA模板，其含有荧光碱基2-氨基嘌呤（2AP），其在单链尾部的-11位置处被取代，所述单链尾部包含非模板链上的核苷酸，在RNA聚合酶-启动子复合物中碱基配对在所述非模板链上被破坏。我们证明，在存在或不存在RNA聚合酶的情况下，2AP取代-11位的A对DNA与DNase I或KMnO 4的可及性没有重大影响，从而证明了在荧光研究中使用含有2AP取代的模板的合理性。在RNA聚合酶存在下观察到2AP荧光发射最大值的蓝移。荧光各向异性衰减研究的结果表明，大约60%的2AP残基在-11位被固定在RNA聚合酶复合物中。该值与确定为与RNA聚合酶形成稳定的肝素抗性复合物的2AP取代模板的分数非常一致。这些结果与-11位的残基紧密结合在酶的疏水口袋中一致。
Strand separation in promoter DNA induced byEscherichia coliRNA polymerase is likely initiated at a conserved A residue at position −11 of the nontemplate strand. Here we describe the use of fluorescence techniques to study the interaction of RNA polymerase with the −11 base. Forked DNA templates were employed, containing the fluorescent base, 2-aminopurine (2AP), substituted at the −11 position in a single-stranded tail comprising the nucleotides on the nontemplate strand at which base pairing is disrupted in an RNA polymerase-promoter complex. We demonstrate that the presence of 2AP instead of an A at position −11 has no major effect on the accessibility of DNA to DNase I or KMnO4in the presence or absence of RNA polymerase, thus justifying the use of templates containing the 2AP substitution in the fluorescence studies. A blue shift of the 2AP fluorescence emission maximum is observed in the presence of RNA polymerase. The results of fluorescence anisotropy decay studies indicate that about 60% of the 2AP residues at −11 are immobilized in an RNA polymerase complex. This value is in good agreement with the fraction of 2AP-substituted templates determined to be in a stable, heparin-resistant complex with RNA polymerase. These results are consistent with the residue at −11 being tightly bound in a hydrophobic pocket of the enzyme.