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Cooperative binding of the Xenopus RNA polymerase I transcription factor xUBF to repetitive ribosomal gene enhancers.

机译:非洲爪蟾RNA聚合酶I转录因子xUBF与重复性核糖体基因增强子的协同结合。

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Upstream binding factor (UBF) is a DNA-binding transcription factor implicated in ribosomal gene promoter and enhancer function in vertebrates. UBF is unusual in that it has multiple DNA-binding domains with homology to high-mobility-group (HMG) nonhistone chromosomal proteins 1 and 2. However, a recognizable DNA consensus sequence for UBF binding is lacking. In this study, we have used gel retardation and DNase I footprinting to examine Xenopus UBF (xUBF) binding to Xenopus laevis ribosomal gene enhancers. We show that UBF has a minimum requirement for about 60 bp of DNA, the size of the short enhancer variant in X. laevis. Stronger UBF binding occurs on the longer enhancer variant (81 bp) and on multiple enhancers linked head to tail. In vivo, Xenopus ribosomal gene enhancers exist in blocks of 10 alternating 60- and 81-bp repeats within the intergenic spacer. In vitro, UBF binds cooperatively to probes with 10 enhancers, with five intermediate complexes observed in titration experiments. This suggests that, on average, one UBF dimer binds every two enhancers. A single UBF dimer can produce a DNase I footprint ranging in size from approximately 30 to about 115 bp on enhancer probes of different lengths. This observation is consistent with the hypothesis that multiple DNA-binding domains or subdomains within UBF bind independently, forming more-stable interactions on longer probes.
机译:上游结合因子(UBF)是一种DNA结合转录因子,与脊椎动物的核糖体基因启动子和增强子功能有关。 UBF之所以与众不同,是因为它具有多个与高迁移率族(HMG)非组蛋白染色体蛋白1和2具有同源性的DNA结合结构域。但是,缺乏可识别的UBF结合DNA共有序列。在这项研究中,我们已经使用凝胶阻滞和DNase I足迹检测了非洲爪蟾UBF(xUBF)与非洲爪蟾核糖体基因增强子的结合。我们表明,UBF具有约60 bp的DNA的最低要求,即短小X. laevis中短增强子的大小。较强的UBF结合发生在较长的增强子变体(81 bp)和头尾相连的多个增强子上。在体内,非洲爪蟾核糖体基因增强子存在于基因间隔区中的10个交替的60和81 bp重复序列中。在体外,UBF与具有10种增强剂的探针协同结合,在滴定实验中观察到5种中间复合物。这表明,平均而言,每两个增强子结合一个UBF二聚体。单个UBF二聚体可在不同长度的增强子探针上产生大小约为30 bp至约115 bp的DNase I足迹。该观察结果与以下假设相一致:UBF内的多个DNA结合结构域或亚结构域独立结合,在更长的探针上形成更稳定的相互作用。

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