Visualizing Shine-Dalgarno independent translation initiation in bacteria by cryo-electron microscopy

通过冷冻电子显微镜观察细菌中 Shine-Dalgarno 独立翻译起始

• 批准号: RGPIN-2019-05799
• 负责人: Ortega, Joaquin
• 金额: $ 3.06万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=715815
• 关键词: Visualizing; Shine; Dalgarno; independent; translation

In all cells, ribosomes translate the genetic code, synthesizing proteins based on the information content of mRNA. The rate of translation varies dramatically, depending on the sequence of the translation initiation region (TIR) of the mRNA, which lies upstream from the start codon. In bacteria, a well-known feature of the TIR is the Shine-Dalgarno (SD) sequence, a purine-rich element located 7-12 nucleotides upstream from the start codon. The SD pairs with the anti-Shine-Dalgarno (ASD), a pyrimidine-rich element at the 3' end of the 16S rRNA (a component of the 30S ribosomal subunit). SD-ASD pairing facilitates start codon recognition and promotes initiation of the protein translation process. As the ASD of 16S rRNA is universally conserved, it has long been presumed that SD-ASD pairing contributes to start codon recognition in all bacteria. However, recent genome sequencing studies have revealed that whole taxonomic groups of bacteria lack SD sequences (and yet retain the conserved ASD). These include the phyla Bacteroidetes. This raises questions: (i) Is there a mechanism that prevents the ASD from pairing to mRNA during initiation? (ii) What are the initiation mechanisms in these organisms? The long-term goal of our research program is to understand the biology of the ribosome. In the research proposed here, we aim to address these questions in the Bacteroidetes, using Flavobacterium johnsoniae as the model organism. The specific objectives are: (1) Determine the structural determinants preventing SD-ASD interaction in the Bacteroidetes initiation complex. (2) Elucidating the role of initiation factors in SD-independent translation initiation. How translation initiation occurs in organism without the wide-spread SD-ASD interaction is an intrinsically interesting biological question. This taxonomic group of Bacteroidetes is large and includes important players of the gut microbiota of mammals. Yet the mechanisms of gene expression remain ill defined. The proposed studies employ a powerful set of complementary approaches, including cryo-electron microscopy to address fundamental questions of translation initiation in Bacteroidetes and may advance the field in a major way. Translation initiation appears to be naturally simplified in the Bacteroidetes, as the complication of SD-ASD pairing is effectively removed. Hence the proposed studies of F. johnsoniae initiation may clarify the roles of other players in translation initiation, such as the secondary structure of the TIR in the mRNA or the function of the ribosomal protein bS1. Such findings would hold broad relevance for all domains of life. The proposed research will also have broader impact. It will provide integrated laboratory research and training programs for graduate and postdoctoral students, including members of underrepresented minorities. The HQP from my laboratory will obtain a unique skill set that will make them competitive in biomedical and pharmaceutical research in Canada.

在所有细胞中，核糖体都会翻译遗传密码，根据 mRNA 的信息内容合成蛋白质。翻译速率变化很大，具体取决于位于起始密码子上游的 mRNA 翻译起始区 (TIR) 的序列。在细菌中，TIR 的一个众所周知的特征是 Shine-Dalgarno (SD) 序列，这是一个富含嘌呤的元件，位于起始密码子上游 7-12 个核苷酸处。 SD 与抗 Shine-Dalgarno (ASD) 配对，ASD 是 16S rRNA（30S 核糖体亚基的一个组成部分）3' 端富含嘧啶的元件。 SD-ASD 配对促进起始密码子识别并促进蛋白质翻译过程的启动。由于 16S rRNA 的 ASD 普遍保守，长期以来人们一直认为 SD-ASD 配对有助于所有细菌的起始密码子识别。然而，最近的基因组测序研究表明，整个细菌分类群都缺乏 SD 序列（但保留了保守的 ASD）。这些包括拟杆菌门。这就提出了问题：（i）是否存在一种机制可以阻止 ASD 在启动过程中与 mRNA 配对？ (ii) 这些生物体的启动机制是什么？我们研究计划的长期目标是了解核糖体的生物学。在此提出的研究中，我们旨在使用约氏黄杆菌作为模式生物来解决拟杆菌门中的这些问题。具体目标是： (1) 确定拟杆菌起始复合物中阻止 SD-ASD 相互作用的结构决定因素。 (2)阐明起始因子在SD独立翻译起始中的作用。 在没有广泛的 SD-ASD 相互作用的情况下，翻译起始如何在生物体中发生是一个本质上有趣的生物学问题。拟杆菌门的这个分类群很大，包括哺乳动物肠道微生物群的重要参与者。然而基因表达的机制仍然不明确。拟议的研究采用了一套强大的补充方法，包括冷冻电子显微镜来解决拟杆菌门翻译起始的基本问题，并可能在很大程度上推动该领​​域的发展。拟杆菌中的翻译起始似乎自然简化，因为 SD-ASD 配对的复杂性被有效消除。因此，对约氏杆菌起始的拟议研究可能会阐明其他参与者在翻译起始中的作用，例如 mRNA 中 TIR 的二级结构或核糖体蛋白 bS1 的功能。这些发现对生活的各个领域都具有广泛的意义。 拟议的研究也将产生更广泛的影响。它将为研究生和博士后学生（包括代表性不足的少数族裔成员）提供综合实验室研究和培训计划。我实验室的 HQP 将获得一套独特的技能，使他们在加拿大的生物医学和药物研究中具有竞争力。