Characterization of ribosomal stalling sequences and their recognition by the RQT quality control factors

核糖体停滞是质量控制机制的标志，其已经进化以防止潜在有毒的新生蛋白质的损害和拯救停滞在异常mRNA上的核糖体。除了mRNA异常外，特定的非最佳密码子对和mRNA的多碱基编码序列也可以引起核糖体停滞。拟议的研究项目旨在发现导致mRNA序列诱导的核糖体停滞的分子机制，并随后控制对这些序列元件的核糖体相关质量控制（RQC）的启动。三个互补的目标将遵循解决关键问题（i）停滞诱导序列对核糖体的具体结构影响是什么，（ii）如何识别停滞的核糖体和（iii）如何触发RQC响应。在所有情况下，将采用单粒子低温电子显微镜（cryo-EM）来获得这些过程的详细机理。首先，我们将使用一个已建立的人工mRNA报告系统，其中含有确认的失速诱导序列。我们将修改这个系统，以编码一系列不同的多元和串联密码子失速诱导序列，以分离特定的失速核糖体。Cryo-EM将用于揭示停滞核糖体的特定结构特征，例如肽基转移酶中心的潜在扰动。接下来，我们将分离和结构表征一种新的RQC触发因子Hel 2与前一步中获得的停滞核糖体复合。这将通过从酵母细胞中分离或通过体外重构来完成。最后，我们计划在存在或不存在Hel 2的情况下获得与新型RQC触发（RQT）复合物（Slh 1-Cue 3-Ykr 023 w）复合的核糖体。在这里，我们将使用与核糖体-Hel 2复合物类似的方法，并使用天然下拉方案来纯化核糖体-RQT复合物以产生合适的冷冻EM样品。同时，我们将从原核或真核表达系统中纯化单个组分（E. coli、S.这些组合的方法将使我们能够对核糖体停滞的机制及其被RQC触发机制识别的重要见解。因此，本项目将提供一个详细的机制基础上的停顿过程所造成的多元和串联密码子mRNA停顿序列。揭示Hel 2如何与核糖体相互作用将揭示其在通过非蛋白酶体泛素化触发RQC中的作用。RQT复合物的后续参与到目前为止就其机制和功能基础而言是完全神秘的，因此我们的目标是提供其在质量控制途径中的结构作用的第一个一瞥。

Ribosomal stalling is a hallmark of quality control mechanisms that have evolved to prevent damage from potentially toxic nascent proteins and rescue of ribosomes stalled on aberrant mRNAs. Besides aberrant mRNA, ribosomal stalling can also be caused by specific non-optimal codon pairs and polybasic encoding sequences of mRNA. The proposed research project aims at discovering the molecular mechanisms, which cause mRNA sequence-induced ribosomal stalling and, subsequently, control initiation of ribosome-associated quality control (RQC) on such sequence elements. Three complementary aims will be followed to address pivotal questions of (i) what the specific structural effects of stalling-inducing sequences on ribosomes are, (ii) how the stalled ribosomes are recognized and (iii) how the RQC response is triggered. In all cases, single-particle cryo-electron microscopy (cryo-EM) will be employed to obtain detailed mechanistic insight into these processes. First, we will use an established artificial mRNA reporter system containing confirmed stalling-inducing sequences. We will modify this system to encode a range of different polybasic and tandem codon stalling-inducing sequences in order to isolate specifically stalled ribosomes. Cryo-EM will be used to reveal specific structural features of stalled ribosomes such as potential perturbations of the peptidyl-transferase center. Next, we will isolate and structurally characterize a novel RQC-triggering factor Hel2 in complex with stalled ribosomes obtained in the previous step. This will be done either by isolation from yeast cells or by in vitro reconstitution. This approach is expected to reveal which common structural features of the specifically stalled ribosomes are recognized by Hel2 in order to trigger the RQC response.Finally, we plan to obtain ribosomes in complex with the novel RQC triggering (RQT) complex (Slh1-Cue3-Ykr023w) in presence or absence of Hel2. Here, we will use a similar approach as for ribosome-Hel2 complexes and use the native pulldown protocol to purify ribosome-RQT complexes to produce suitable cryo-EM samples. In parallel, we will purify the individual components from prokaryotic or eukaryotic expression systems (E. coli, S. cerevisiae, insect or human cell culture) and reconstitute them with stalled ribosomes.These combined approaches will allow us to gain important insights into the mechanisms of ribosomal stalling and its recognition by the RQC-triggering machinery. This project will, thus, provide a detailed mechanistic basis of the stalling process caused by polybasic and tandem codon mRNA stalling sequences. Revealing how Hel2 interacts with the ribosome will shed light on its role in triggering RQC by non-proteasomal ubiquitination. The subsequent engagement of the RQT complex is entirely enigmatic so far with respect to its mechanistic and functional basis, and we therefore aim to provide a first glimpse at its structural role in the quality control pathway.