Single Molecule Dynamics of mRNA Translation

mRNA 翻译的单分子动力学

• 批准号: 8708889
• 负责人: BARRY S. COOPERMAN
• 金额: $ 29.82万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8708889
• 关键词: Acquired Immunodeficiency Syndrome; Address; Affect; Amino Acids; Amino Acyl Transfer RNA; Antibiotics; Bacterial Infections; Binding; Biological Assay; C-terminal; CCL4 gene; Cell physiology; Cells; Chemicals; Code; Codon Nucleotides; Complement; Complex; Coupling; Development; Discrimination; Elements; Energy Transfer; Erythromycin; Escherichia coli; Eukaryotic Cell; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Genes; Genetic Translation; Genome; Goals; Green Fluorescent Proteins; Human; Indium; Individual; Kinetics; Knowledge; Label; Length; Life; Ligand Binding; Malignant Neoplasms; Mammalian Cell; Measures; Membrane; Messenger RNA; Microscope; Microscopy; Modeling; Molecular Chaperones; Monitor; Motion; Neomycin; Organism; Peptides; Peptidyltransferase; Performance; Post-Translational Protein Processing; Process; Proline-Specific tRNA; Protein Biosynthesis; Proteins; Regulation; Relative (related person); Reporter; Resistance; Ribosomes; Signal Transduction; Site; Structure; Techniques; Testing; Time; Transfer RNA; Transfer RNA Aminoacylation; Translations; Variant; Virus Replication; base; combat; insight; pathogen; programs; protein aminoacid sequence; protein expression; protein folding; research study; ribosomal protein L1; single molecule; single-molecule FRET

Regulation of translation rate is of crucial importance for such downstream co-translational pro- cesses as protein folding and modification, ligand binding, oligomerization, and interactions with chaperones and membranes. Although specific elements that modulate translation rate are well known, at present there is virtually a complete lack of results describing how such elements quantitatively affect translation rate. Our overall goal is to address this lack by quantifying the modulation of the rate of translation by E. coli ribosomes of specific mRNA and nascent peptide sequences, and to elucidate the mechanisms of such modulation. We use an approach coupling Total Internal Reflection Fluorescence Microscopy (TIRFM) with single molecule Fluorescence Resonance Energy Transfer (smFRET). Fluorescent reporter and smFRET pairs are introduced specifically into both aminoacyl tRNAs and ribosomal proteins L1 and L11, which are proximal to the tRNA E-site and A-site, respectively. The availability of these fluorescent-labeled compo- nents of the protein synthesis machinery permits observation on single ribosomes and in real time of FRET interactions monitoring aminoacyl-tRNA binding to the ribosomal A-site, interme- diate motions of tRNAs in adjacent sites, translocation of the tRNAs to the P- and E-sites, and release of discharged tRNA from the E-site. Single molecule assays have been developed both for short model mRNAs that emphasize pairing with fluorescent tRNAs and for mRNAs coding full-length proteins with the natural complement of all amino acids. Full expression of a rapidly maturing variant of green fluorescent protein, Emerald GFP (EmGFP), is signaled by fluores- cence of single EmGFP molecules in the TIRF microscope. Together, these approaches allow both discrimination of individual steps within the elongation cycle and continuous kinetic profiles of mRNA translation, providing unique insights into the regulation of translation rates in prokary- otic protein synthesis not accessible from other techniques. Our results will allow us to quantify the effects of modulatory elements and to determine if new elements and synergistic or antago- nistic effects are operative. Crucial for the correct interpretation of our results is the performance of control experiments demonstrating that the rate effects we measure result from introduction of the intended modulating element, rather than from involvement of one or more other ele- ments. We have 3 Specific Aims that focus on determining how the rhythm of protein synthesis is modulated by: 1. Codon usage and codon pair usage; 2. Pause-inducing nascent peptides bound within the peptidyl transferase center of the ribosomes and the peptide exit tunnel; and 3. Pausing elements present in combination. Overall, our program of studying several pause ele- ments at the full protein expression level and within individual elongation cycles will define the magnitudes and detailed mechanisms of translation regulation.

翻译速率的调节对于这种下游共翻译亲和过程至关重要。 蛋白质折叠和修饰、配体结合、寡聚化以及与 伴侣和膜。尽管调节翻译速度的特定元素很好 众所周知，目前几乎完全缺乏描述这些元素如何 定量地影响翻译率。我们的总体目标是通过量化来解决这一不足 大肠杆菌核糖体对特定 mRNA 和新生肽翻译速率的调节 序列，并阐明这种调制的机制。我们使用耦合方法 单分子荧光全内反射荧光显微镜 (TIRFM) 共振能量转移（smFRET）。引入荧光报告基团和 smFRET 对 特异地进入氨酰 tRNA 和核糖体蛋白 L1 和 L11，它们是近端的 分别连接至 tRNA E 位点和 A 位点。这些荧光标记成分的可用性 蛋白质合成机器的结构允许对单个核糖体进行观察 FRET 相互作用的时间监测氨酰基-tRNA 与核糖体 A 位点的结合，中间体 tRNA 在相邻位点的运动、tRNA 易位至 P 位点和 E 位点，以及 从 E 位点释放排出的 tRNA。单分子检测已被开发出来 对于强调与荧光 tRNA 配对的短模型 mRNA 以及编码 mRNA 具有所有氨基酸天然补充的全长蛋白质。充分表达了快速 绿色荧光蛋白 Emerald GFP (EmGFP) 的成熟变体由荧光信号发出 TIRF 显微镜中单个 EmGFP 分子的存在。这些方法共同允许 区分伸长周期内的各个步骤和连续动力学曲线 mRNA 翻译，为原核翻译速率的调节提供独特的见解 其他技术无法实现耳蛋白合成。我们的结果将使我们能够量化 调节元素的作用并确定新元素是否具有协同或拮抗作用 本质效应正在发挥作用。对于正确解释我们的结果至关重要的是性能 控制实验证明我们测量的速率效应是由引入引起的 预期的调节元素，而不是来自一种或多种其他元素的参与 评论。我们有 3 个具体目标，重点是确定蛋白质合成的节律 受以下因素调节： 1. 密码子使用和密码子对使用； 2. 暂停诱导新生肽 结合在核糖体的肽基转移酶中心和肽出口通道内；和 3. 暂停组合中存在的元素。总的来说，我们研究几个暂停元素的计划 在完整的蛋白质表达水平和单个延伸周期内的变化将定义 翻译调节的程度和详细机制。