Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking.
Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking.
复制标题
通过光亲和交联研究转录延伸复合物中酵母 RNA 聚合酶 II 亚基的拓扑结构。
DOI：
10.1021/bi0014249
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Burgess,RR
中科院分区：
文献类型：
--
作者：
Wooddell,CI;Burgess,RR
用光亲和交联法鉴定了酿酒酵母RNA聚合酶II(RNAP II)在转录延伸过程中靠近DNA的亚基。在没有转录因子的情况下，RNAP II将转录一个双链DNA片段，其中包含一个脱氧胞苷的3‘-延伸，即一个“拖尾模板”。我们设计了一个DNA模板，允许RNAP在转录反应中因省略CTP而停滞不前之前转录76个碱基。这个摊位定位了DNA模板上的RNAP，并允许我们沿着DNA绘制RNAP亚基的图谱。合成了DNA类似物5-[N-(对叠氮苯甲酰基)-3-氨基烯丙基]-dUTP(N3RdUTP)[Bartholomew，B.，Kosa vetis，G.A.，Braun，B.R.和Geiduschek，E.P.(1990)EMBO J.9,2197−205]，并在DNA的模板或非模板链中，在摊位的上游或下游的特定位置酶结合到DNA中。放射性核苷酸被定位在可光激活的核苷酸旁边，通过短暂的紫外线照射进行交联，将放射性标记从DNA转移到RNAP亚基上。除了N3RdUTP具有来自尿苷碱基的光反应叠氮基9ä外，我们使用光亲和交联剂5N3dUTP将叠氮基团直接连接在尿苷环上，以确定在多个亚基交联处距离DNA最近的RNAP II亚基。在依赖转录的交联反应中，RPB1、RPB2和RPB5与N3RdUTP发生交联。在5N3dUTP中，只有RPB1和RPB2发生了交联。在某些情况下，RPB3、RPB4和RPB7是交联的。根据这项拓扑研究中获得的信息，我们开发了酵母RNAP II在转录延伸复合体中的模型。
The subunits ofSaccharomyces cerevisiaeRNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking. In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3‘-extension of deoxycytidines, a “tailed template”. We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction. This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA. The DNA analogue 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N3RdUTP) [Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990)EMBO J.9, 2197−205] was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA. Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits. In addition to N3RdUTP, which has a photoreactive azido group 9 Å from the uridine base, we used the photoaffinity cross-linker 5N3dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked. In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N3RdUTP. With 5N3dUTP, only RPB1 and RPB2 were cross-linked. Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked. From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex.