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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=688570
• 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，核糖体翻译遗传密码，根据mRNA的信息内容合成蛋白质。翻译的速率变化很大，这取决于mRNA的翻译起始区（TIR）的序列，它位于起始密码子的上游。在细菌中，TIR的一个众所周知的特征是Shine-Dalgarno（SD）序列，一种位于起始密码子上游7-12个核苷酸的富含嘌呤的元件。SD与抗Shine-Dalgarno（ASD）配对，ASD是16 S rRNA（30 S核糖体亚基的一个组分）3'端的富含嘧啶的元件。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16 S rRNA的ASD是普遍保守的，长期以来一直认为SD-ASD配对有助于所有细菌中的起始密码子识别。然而，最近的基因组测序研究表明，细菌的整个分类群缺乏SD序列（但保留保守的ASD）。其中包括拟杆菌门。这就提出了一个问题：（i）是否有一种机制可以阻止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。在这里提出的研究中，我们的目标是解决这些问题的拟杆菌，使用黄杆菌约翰逊作为模式生物。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非依赖性翻译起始中的作用 *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。拟杆菌属的这一分类群很大，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互补方法，包括冷冻电子显微镜来解决拟杆菌中翻译起始的基本问题，并可能以主要方式推进该领域。翻译起始似乎在拟杆菌中自然简化，因为SD-ASD配对的复杂性被有效地去除。因此，F.约翰逊菌的起始可以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bS 1的功能。这些发现将对生活的所有领域具有广泛的意义。***它将为研究生和博士后学生提供综合实验室研究和培训计划，包括代表性不足的少数民族成员。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制药研究中具有竞争力。