Functional characterization of the chaperone network connected to the eucaryotic ribosome

现在认识到，核糖体结合的新生肽可以与多肽出口通道的组分相互作用，获得二级结构，并将信息传递到核糖体的远端位点。然而，其潜在的机制还不完全清楚，特别是在真核系统中。该项目的目的是了解真核生物核糖体，新生链和核糖体结合蛋白质生物合成因子（RPB）之间的相互作用如何影响蛋白质生物合成的早期步骤。我们将重点分析核糖体蛋白Rpl17，Rpl4和Rpl39，它们与真核生物中的隧道内部接触。将采用Rpl17和Rpl4内的随机诱变来产生一组条件致死酵母突变体。将在体内表征表达Rpl17、Rpl4的突变形式或缺乏Rpl39的酵母菌株。我们将重点关注突变株中的蛋白质靶向和折叠缺陷。一系列的生物化学方法将被用来比较新生链的野生型和突变体核糖体的结合的命运。首先，我们将确定核糖体隧道蛋白影响新生链折叠的机制。其次，我们将分析新生链折叠是否影响主要的核糖体结合蛋白质生物合成因子SRP、NAC和Ssb的功能。特别地，我们计划跟踪信号锚序列从隧道到SRP的转移。分析将阐明信号锚序列的核糖体内折叠是否是SRP依赖性靶向的先决条件，以及什么确切地定义了SRP依赖性底物。最后，我们将采用该系统来测试的假设，新生链可以促进核糖体结合位点内的结构变化的RPB从核糖体隧道内。

It is now recognized that ribosome-bound nascent peptides can interact with components of the polypeptide exit tunnel, acquire secondary structure, and transmit information to distant sites of the ribosome. The underlying mechanisms, however, are only incompletely understood, specifically in the eukaryotic system. Aim of the project is to understand how the interplay between components of the eukaryotic ribosome, nascent chains, and ribosomebound protein biogenesis factors (RPBs) affects the early steps of protein biogenesis. We will focus on the analysis of the ribosomal proteins Rpl17, Rpl4, and Rpl39, which contact the interior of the tunnel in eukaryotes. Random mutagenesis within Rpl17 and Rpl4 will be employed to generate a set of conditionally lethal yeast mutants. Yeast strains expressing mutant versions of Rpl17, Rpl4, or lacking Rpl39 will be characterized in vivo. We will focus on protein targeting and folding defects in the mutant strains. An array of biochemical methods will then be employed to compare the fate of nascent chains bound to wild type and mutant ribosomes. First, we will determine by what mechanism the ribosomal tunnel proteins affect nascent chain folding. Second, we will analyze if nascent chain folding impacts on the function of the major ribosome-bound protein biogenesis factors, SRP, NAC, and Ssb. In particular we plan to trace the transfer of a signal anchor sequence from the tunnel to SRP. The analysis shall unravel if intraribosomal folding of a signal anchor sequence is a prerequisite for SRP-dependent targeting, and what exactly defines SRPdepended substrates. Finally, we will employ the system to test the hypothesis that nascent chains can facilitate structural changes within the ribosomal binding site(s) of RPBs from within the ribosomal tunnel.