【作者】 孙奇；

【导师】 叶克穷；

【作者基本信息】 清华大学 ， 生物学， 2017， 博士

【摘要】 真核生物的核糖体发生是一个极其复杂而精密的过程,约有两百多个组装因子参与其中。其中90S核糖体前体,又称小亚基加工体,是最早形成的一个巨大复合物,完整的90S约有5兆道尔顿的分子量。利用冷冻电镜单颗粒技术,我们解析了接近完整的酵母90S的结构,分辨率从8.7?至4.5?。其中在Mtr4敲除的样品中得到了4.5?最高分辨率的样品。结合已知晶体结构、同源模型和化学交联质谱数据,我们搭建了大部分蛋白因子的结构模型,包括36种组装因子和19种核糖体小亚基蛋白;手动搭建了5’ETS RNA的10个双螺旋区中的8个;18S RNA的部分则是在成熟18S的基础上修正。本文按5’ETS蛋白(UTP-A、UTP-B、U3snoRNP)、5’ETS RNA、18S 5’结构域、central结构域、3’major结构域、3’minor结构域的顺序,分区域描述90S的结构。在我们的90S模型中可以看到UTP-A、UTP-B以相似的α螺旋束形成的异源四聚体;U3 RNA与5’ETS RNA和18S有四个互补位点,其中最后一个位点是首次发现;18S的各个结构域都已经部分形成,但是相对位置与成熟40S中存在较大差别。测试了90S加工过程中两个RNA解旋酶Dhr1和Mtr4对90S结构的影响。在Dhr1敲除的样品中,我们观察到了90S的三种状态。除了正常的状态以外,还有两种5’ETS RNA被降解的两种状态,并结合生化和质谱的证据,确定了Mtr4募集exosome组分在降解5’ETS RNA方面的直接作用。在5’ETS RNA被降解后,90S仍然可以保持整体轮廓,尤其是5’ETS蛋白仍然结合在90S上,证明了5’ETS RNA降解和90S的解体是两个相对独立的过程,90S至40S的加工步骤要比现有的模型更加复杂。更多还原

【Abstract】 The ribosome biogenesis of eukaryotes is an extremely complex and sophisticated process involving about two hundred assembly factors.90S-preribosome,or small subunit processome,is the earliest gigantic complex formed during ribosome biogenesis,with about 5 megadaltons.Using cryo-EM single particle reconstruction,we solved three structures of the nearly complete yeast 90 S with the resolution from 8.7 ? to 4.5 ?,among which,the highest resolution,4.5 ?,was obtained from the Mtr4 knock-out sample.Combined with known crystal structures,homology models and chemical cross-linking coupled with mass spectrometry,we build the model of most protein factors,including 36 assembly factors and 19 small subunit proteins.Eight of the ten double helixes of 5’ ETS RNA were constructed manually.The 18 S RNA model was modified on the basis of mature 18 S.This dissertation will describe the structure of 90 S in the sequence of 5’ETS protein(UTP-A,UTP-B,U3 snoRNP),5’ ETS RNA,18 S 5’ domain,central domain,3’ major domain,3’ minor domain,respectively.In our model of the 90 S,we observed the similar heterogeneous tetramers consisted of α-helix bundles formed in UTP-A and UTP-B subcomplex.U3 RNA could form four complementary sites with 5’ ETS RNA and 18 S RNA.The last complementary site was reported at first time.Each of four domains of 18 S RNA has partially formed in the 90 S,but the relative position among them was quite different from that in mature 40 S.We tested the impact of Dhr1 and Mtr4,two RNA helicases involved in 90 S processing,on the structure of 90 S.In the Dhr1 knock-out sample,we observed three states of 90 S.Apart from the normal 90 S,two other states were without 5’ ETS RNA.Combined with the evidences of biochemistry and mass-spectrometry,we determined the function of Mtr4 in recruiting exosome components and degrading 5’ ETS RNA.After the degradation of 5’ ETS RNA,the 90 S could still maintain the overall shape.Surprisingly,the 5’ ETS proteins were still bound to the 90 S,demonstrating that 5’ ETS RNA degradation and disintegration of the 90 S are two relatively independent processes.The processing steps transiting the 90 S to the 40 S are more complex than existing model.更多还原