ORIENTATION AND TOPOGRAPHY OF RNA POLYMERASE-III IN TRANSCRIPTION COMPLEXES
ORIENTATION AND TOPOGRAPHY OF RNA POLYMERASE-III IN TRANSCRIPTION COMPLEXES
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DOI：
10.1128/mcb.13.2.942
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发表时间：
1993-02-01
影响因子：
5.3
通讯作者：
GEIDUSCHEK, EP
中科院分区：
文献类型：
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作者：
BARTHOLOMEW, B;DURKOVICH, D;GEIDUSCHEK, EP
光交联方法已用于绘制酿酒酵母 RNA 聚合酶 (Pol) III 亚基相对于二元（起始前）和三元（RNA 延伸）转录复合物中 DNA 的图谱。含有转录因子和 Pol III 的复合物已组装在酿酒酵母 SUP4 tRNA(Tyr) 基因探针上，该探针在不同的指定位置含有光活性核苷酸 5-[N-(对叠氮基苯甲酰基)-3-氨基烯丙基]-dUMP。这些复合物受辐射后形成共价 DNA-蛋白质连接，并且已鉴定出与 SUP4 tRNA 基因中各个位置交联的 Pol III 亚基。 RNA Pol III 交联已被证明需要 tRNA 基因的 Box B 下游启动子元件以及转录因子 tFIIIB 的存在。通过证明特定的 Pol III 亚基移出上游放置的光活性核苷酸的范围，而其他亚基移入下游放置的光活性核苷酸的范围，作为启动和延长 RNA 链的结果，提供了进一步的特异性证明。 Pol III 的结合和特定放置也已被证明需要 TFIIIB 的 B' 和 B'' 组件。九个 Pol III 亚基从 SUP4 tRNA 基因非转录链的不同位置交联。在二元转录复合物中，两个最大的 Pol III 亚基可在整个 DNase I 足迹上进行光交联。 27-和 34-kDa Pol III 亚基也沿着 DNA 相对延伸；其上游投影使 34 kDa 亚基成为与 TFIIIB 相互作用的候选者，而 27 kDa 亚基可从 Pol III 结合位点的前缘进行光交联。几个亚基，包括二元转录复合物中的 82 kDa 和 53 kDa 亚基，其交联可及性相对局部化。多个 Pol III 亚基可通过位于阻滞三元转录复合物的转录泡中间的单个光活性核苷酸进行特异性交联。有人认为，这种精确放置的转录复合体包含结构状态的动态整体，而不是单个完美约束的实体。
A photo-cross-linking method has been used to map the subunits of Saccharomyces cerevisiae RNA polymerase (Pol) III with respect to DNA in binary (preinitiation) and ternary (RNA-elongating) transcription complexes. Transcription factor- and Pol III-containing complexes have been assembled on S. cerevisiae SUP4 tRNA(Tyr) gene probes containing the photoactive nucleotide 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP in different specified positions. Covalent DNA-protein linkages form upon irradiation of these complexes, and the Pol III subunits that are cross-linked to individual positions in the SUP4 tRNA gene have been identified. RNA Pol III cross-linking has been shown to require the box B downstream promoter element of the tRNA gene and the presence of transcription factor tFIIIB. Further proof of specificity has been provided by demonstrating that particular Pol III subunits move out of the range of upstream-placed photoactive nucleotides, and that others move into the range of downstream-placed photoactive nucleotides, as a consequence of initiating and elongating RNA chains. Binding and specific placement of Pol III have also been shown to require both the B' and the B'' components of TFIIIB. Nine Pol III subunits are cross-linked from different positions of the SUP4 tRNA gene's nontranscribed strand. In binary transcription complexes, the two largest Pol III subunits are accessible to photo-cross-linking over the entire stretch of the DNase I footprint. The 27- and 34-kDa Pol III subunits are also relatively extended along DNA; its upstream projection makes the 34-kDa subunit a candidate for interaction with TFIIIB, while the 27-kDa subunit is accessible to photo-cross-linking from the leading edge of the Pol III binding site. Several subunits, including the 82- and 53-kDa subunits in binary transcription complexes, are relatively localized in their accessibility to cross-linking. Multiple Pol III subunits are accessible to specific cross-linking from a single photoactive nucleotide in the middle of the transcription bubble of an arrested ternary transcription complex. It is suggested that this precisely placed transcription complex comprises a dynamic ensemble of structural states rather than a single perfectly constrained entity.