Molecular mechanisms of translation termination in bacteria

翻译终止是所有单细胞和多细胞生物体的一个基本生物学过程，它确保了由称为核糖体的分子机器制造的所有蛋白质的长度都由编码它们的基因严格定义。这一过程由称为释放因子的蛋白质介导，其促进新合成的多肽产物从核糖体释放。必须严格控制释放，以防止可能对细胞无功能或甚至有毒的截短或延伸多肽的积累。本项目的目标是阐明释放因子在终止蛋白质合成的基本生物学过程中的分子机制。该项目将对我们在分子水平上理解生物学的进步产生广泛的影响。 最终，待确定的核糖体机器的机械细节可以揭示靶向细菌核糖体的抗生素开发的新途径。 此外，该项目还将培训本科生和研究生掌握结构生物学的强大技术，并向高中生介绍结构和分子生物学前沿的研究。所有释放因子（RF）共享普遍保守的三肽序列基序Gly-Gly-Gln（GGQ），其对于在核糖体的活性位点（肽基转移酶中心）引发水解活性至关重要。尽管之前进行了大量的生物化学和结构研究，但释放因子如何促进肽基转移中心肽基-tRNA水解的确切分子机制仍然难以捉摸。因此，该项目的总体目标是揭示RF介导的肽释放的这种尚未确定的催化机制，并回答该领域长期存在的基本问题：释放因子如何促进核糖体中肽基-tRNA的水解，以及普遍保守的GGQ三肽基序对此过程的贡献是什么？作为这项工作的一部分，将确定终止前70 S-RF复合物的多个晶体结构。这些结构需要制备70 S-RF复合物，并使用不可水解的酰胺连接肽基-tRNA进行工程改造。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Translation termination - an essential biological process in all unicellular and multicellular organisms - ensures that all proteins manufactured by the molecular machine known as the ribosome have the lengths strictly defined by the genes that code for them. This process is mediated by proteins know as Release Factors, which promote release of newly synthesized polypeptide product from the ribosome. Release must be tightly controlled to prevent the accumulation of truncated or extended polypeptides that can be non-functional or even toxic to the cell. The goals of the current project are to elucidate the molecular mechanism implemented by the Release Factors during the fundamental biological process of termination of protein synthesis. This project will have a broad impact on the advancement of our understanding of biology at the molecular level. Ultimately, the mechanistic details of the ribosomal machinery to be determined can reveal new avenues for the development of antibiotics that target the bacterial ribosome. In addition, the project will train both undergraduate and graduate students in powerful techniques in structural biology and introduce high school students to research at the forefront of structural and molecular biology. All Release Factors (RF) share a universally conserved tripeptide sequence motif Gly-Gly-Gln (GGQ) that is critical for eliciting hydrolytic activity at the active site of the ribosome, the peptidyl transferase center. Despite a number of previous biochemical and structural studies, the exact molecular mechanism of how Release Factors promote hydrolysis of peptidyl-tRNA at the peptidyl transfer center remains elusive. Thus, the overall goal of the proposed project is to uncover this yet undefined catalytic mechanism of RF-mediated peptide release and answer the long-standing fundamental questions in the field: how do release factors promote hydrolysis of the peptidyl-tRNA in the ribosome and what is the contribution of the universally conserved GGQ tripeptide motif to this process? As part of this work, multiple crystal structures of pre-termination 70S-RF complexes will be determined. These structures require the preparation of 70S-RF complexes trapped in the pre-termination state with the use non-hydrolyzable amide-linked peptidyl-tRNAs that will be engineered.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.