Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation
Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation
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DOI：
10.1038/nsmb.1458
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发表时间：
2008-08-01
影响因子：
16.8
通讯作者：
Cramer, Patrick
中科院分区：
文献类型：
--
作者：
Brueckner, Florian;Cramer, Patrick
为了研究RNA聚合酶II如何在核苷酸掺入后易位，我们制备了延伸复合物晶体，其中易位前和易位后状态相互转换。用抑制剂α-鹅膏蕈碱浸泡晶体将延伸复合物锁定在一个新的状态，该状态在3.4埃分辨率下进行了细化，并被鉴定为可能的易位中间体。进入活性位点的DNA碱基占据中心桥螺旋上方的“预模板化”位置，其被移位并封闭模板化位置。触发环中的亮氨酸残基在移位的桥螺旋处形成楔形，但在核苷酸掺入期间移动13埃以关闭活性位点。我们的研究结果支持一个布朗棘轮机制，涉及摆动的触发环之间的开放，楔形和关闭的位置，并建议α-鹅膏蕈碱损害核苷酸掺入和易位通过捕获触发环和桥螺旋。
To study how RNA polymerase II translocates after nucleotide incorporation, we prepared elongation complex crystals in which pre- and post-translocation states interconvert. Crystal soaking with the inhibitor alpha-amanitin locked the elongation complex in a new state, which was refined at 3.4-angstrom resolution and identified as a possible translocation intermediate. The DNA base entering the active site occupies a 'pretemplating' position above the central bridge helix, which is shifted and occludes the templating position. A leucine residue in the trigger loop forms a wedge at the shifted bridge helix, but moves by 13 angstrom to close the active site during nucleotide incorporation. Our results support a Brownian ratchet mechanism that involves swinging of the trigger loop between open, wedged and closed positions, and suggest that alpha-amanitin impairs nucleotide incorporation and translocation by trapping the trigger loop and bridge helix.