Molecular characterisation and function of nucleolar and nuclear protein complexes involved in sequential maturation steps of ribosomal precursor particles

我们从酵母核仁（SNOS为亚核仁结构）中分离出一个大的蛋白质复合物。鉴定的蛋白包括RNA聚合酶I （pol I）、pol I转录因子TBP、Rrn3p、Rrn10p和Reb1p以及rRNA加工因子Nop1p、Cbf5p、Nhp2p和Rrp5p。该复合体支持rRNA的准确转录、终止和修饰，这表明存在一个产生成熟rRNA的合作网络。因为大多数组件(？30)在尚未确定的大型分离复合体中，MALDI质谱法应该作为鉴定其他（新的）核蛋白的工具。在我们已经在亚核仁结构中确定的成分中，有两种新的蛋白质，它们是真核蛋白家族的成员，基于它们对某些亚结构域的惊人保护。由于其中一个保守结构域预测了与DNA的选择性结合，因此将分析蛋白质与DNA，特别是与rDNA单元结构域的相互作用。亚核仁结构的两个进一步确定的因子显然参与转录rDNA单元。它们最初都被认为是有效转录mRNA所必需的。我们将结合生物化学、遗传和细胞生物学的方法来分析鉴定的（新）蛋白质在rRNA的合成和加工中的作用，以及它们在构建核仁结构中的可能参与。

We have isolated a large protein complex from the yeast nucleolus (SNOS for subnucelolar structure). Among the identified proteins were RNA polymerase I (pol I), pol I transcription factors TBP, Rrn3p, Rrn10p and Reb1p and rRNA processing factors Nop1p, Cbf5p, Nhp2p and Rrp5p. The complex supports accurate transcription, termination and modification of rRNA suggesting the existence of a cooperative network to produce mature rRNA. Since most of the compontents (?30) within the large isolated complex remain to be determined, MALDI mass spectrometry should serve as a tool to identify other (novel) nucleolar proteins. Among the components that we have already identified within the subnucleolar structure are two novel proteins which are members of a eukaryotic protein family based on their striking conservation of certain subdomains. Since one of the conserved domain predicts selective binding to DNA the interaction of the proteins with DNA, especially with domains of the rDNA unit will be analysed. Two further identified factors ot the subnucleolar structure are obviously involved in transcribing the rDNA unit. They were both originally described to be required to transcribe efficiently mRNA. We will use a combination of biochemical, genetic and cell biological methods to analyse the roles of the identified (novel) proteins in synthesis and processing of rRNA and their possible involvement in building up the nucleolar structure.