酿酒酵母的核糖体由79个蛋白质和4条RNA构成，其组装过程非常复杂，需要约200个反式作用因子和许多snoRNA的参与。这个过程始于核糖体前体RNA(pre-rRNA)在核仁中的转录。pre-rRNA在转录过程中，与约70个组装因子、U3、U14和snR30 snoRNA和约20个核糖体蛋白以逐步和动态的方式组装成一个巨大的90S小亚基核糖体前体。在pre-rRNA的A0、A1和A2位点发生切割之后，90S颗粒转化为pre-40S核糖体前体。pre-40S在细胞质中进一步成熟为40S核糖体小亚基。UTPA复合体是90S核糖体前体的关键组成部分，也是第一个被招募到pre-rRNA上的复合物。UTPA复合物由7种蛋白质(Utp4、 Utp5、Utp8、Utp9、Utp10、Utp15和Utp17)组成。Utp10经预测全部由α螺旋构成，其他UTPA蛋白都含有一个或两个WD结构域。 本文采用X射线晶体学、生物化学和酵母遗传学等方法研究了UTPA复合物的结构和功能。我们运用酵母双杂交和体外下拉的方法确定了每个UTPA蛋白相互作用的结构域。Utp5、Utp15、Utp8和Utp9通过各自的C端结构域(CTD)相互作用。Utp4结合Utp5和Utp8的C端尾部氨基酸序列。Utp17通过一段C端的短肽与Utp10的N结构域相互作用。这些相互作用和90S冷冻电镜结构一致，也帮助了90S冷冻电镜密度中UTPA模型的搭建。 我们测定了嗜热毛壳菌Utp10的N和M结构域分辨率分别为2.5Å和3.2Å,的晶体结构。Utp10的N和M结构域都是由两个类似HEAT α螺旋亚结构域构成，亚结构域以几乎直角的方式排列。我们测定了嗜热毛壳菌Utp10 N结构域和Utp17 C端小肽复合物的2.6Å分辨率的晶体结构。结构显示，Utp17 C端小肽折叠成α螺旋发夹结构，主要是通过疏水作用结合在Utp10 N结构域的内表面。为了研究Utp17和Utp10相互作用的功能，我们构建了条件性表达Utp17的酵母菌株，并通过质粒表达Utp17突变体。在Utp17缺失或突变的情况下，我们分析了酵母生长表型，用Northern分析rRNA的加工，用半定量质谱方法分析了90S中组装因子的变化。在所有条件下，敲除Utp17都是致死的，并阻碍了Utp10组装到UTPA复合体和90S的组装。切除Utp17用于结合Utp10 N的C端小肽基本不影响酵母在20℃的生长，但严重抑制酵母在30和37℃的生长。破坏Utp10和Utp17的相互作用会阻碍pre-rRNA在A0、A1和A2位点的加工，18S rRNA的生成和Utp10组装到UTPA复合物。在没有与Utp17相互作用的情况下，Utp10仍然可以组装到90S上，这可能是Utp10与90S的其他成分相互作用介导的。但是这种情况下组装的90S颗粒的量很少，且性质不稳定。 我们测定了嗜热毛壳菌Utp15 C端结构域(CTD)2.7Å分辨率的晶体结构。 Utp15 CTD全部由α螺旋组成，并形成同源二聚体结构。同源二聚体结构似乎模拟了生理条件下Utp15和Utp5的CTD形成的异源二聚体复合物。 总之，我们的研究有助于深入理解UTPA复合物的结构和组装方式以及Utp10和Utp17相互作用在90S组装中的功能。 关键词：90S核糖体前体，核糖体组装，晶体结构，RNA蛋白质复合物

 The ribosome from the yeast Saccharomyces cerevisiae is assembled from 79 proteins and 4 RNAs through a complicated process that requires ～ 200 trans-acting proteins and many snoRNAs. The process begins with the transcription of a precursor ribosomal RNA (pre-rRNA) in the nucleolus. The pre-rRNA is co-transcriptionally assembled with ～70 assembly factors, the U3, U14 and snR30 snoRNAs and ～20 ribosomal proteins in a stepwise and dynamic manner into a large 90S small subunit pre-ribosome. The 90S particle is transformed into the pre-40S ribosome after cleavage at the AO, A1 and A2 sites of pre-rRNA. The pre-40S develops into mature 40S ribosome in the cytoplasm. The UTPA complex is a key component of 90S pre-ribosome and the first recruited to the pre-rRNA. The UTPA complex is composed of seven proteins (Utp4, Utp5, Utp8, Utp9, Utp10, Utpl5 and Utp17). Except for Utp10, which is predicted to be all helical, all UTPA proteins contain one or two WD domains. This thesis studies the structure and function of the UTPA complex with X-ray crystallography, biochemistry and yeast genetics approaches. We have mapped the interacting domains of each UTPA proteins with yeast two-hybrid and pull down assay. Utp5, Utpl5, Utp8 and Utp9 interact with each other via their C-terminal domains (CTD). Utp4 binds the C-terminal tails of Utp5 and Utp8. A C-terminal peptide of Utp17 interacts with the N domain of Utp10. The interaction map is consistent with and facilitates the building of the structure of UTPA in the cryo-EM density map of 90S. We have determined the crystal structures of the N and M domain of Utp10 from Chaetomium thermophilum at 2.5 and 3.2 Å resolution, respectively. The N and M domains of Utp10 are each composed of two HEAT-like helical repeat units that are packed at nearly right angles. We also determined the crystal structure of the Utp10 N domain bound to a C-terminal peptide of Utp17 using C. thermophilum proteins at 2.6 Å resolution. The structure reveals that the C-terminal peptide of Utp17 folds into a helical hairpin and binds at the inner surface of the Utp10 N domain with mainly hydrophobic interactions. To study the functional role of Utp17 and Utp10 interaction, we constructed a S. cerevisiae strain where the genomic Utp17 gene is conditionally expressed and a Utp17 mutant is expressed from plasmid. Upon depletion or mutating of Utp17, we analyzed yeast growth, rRNA processing with northern blot and protein composition of 90S with semi-quantitative mass spectrometry. Depletion of Utp17 is lethal at all conditions and blocks the assembly of Utp10 to the UTPA complex and assembly of 90S. Deletion of the C-terminal Utp10-binding region of Utp17 is largely tolerated at 20 ℃, but severely inhibits yeast growth at 30 and 37 ℃. Disruption of the Utp10 and Utp17 interaction blocks rRNA processing at AO, A1 and A2 sites, 18S rRNA production and assembly of Utp10 to the UTPA complex. Surprisingly, Utp10 can be still assembled into 90S in the absence of Utp17 interaction, likely via interactions with other components of 90S. However, the 90S particle assembled with the Utp17 mutant is scarce and unstable. We determined the crystal structure of the C-terminal domain (CTD) of C. thermophilum Utpl5 at 2.7 A resolution, which reveals an all-helical fold forming a homodimer. The homodimeric structure appears to mimic the physiological heterodimeric complex formed by the CTDs of Utp15 and Utp5. In sum, our study provides insight into the structure and organization of UTPA complex and the functional role of Utp17 and Utp10 interaction for 90S assembly. Keywords: 90S pre-ribosome, ribosome assembly, crystal structure, RNA-protein complex