Effects of Translational Pausing Elements on the Rhythm of Protein Synthesis

翻译暂停元件对蛋白质合成节律的影响

• 批准号: 8782964
• 负责人: AMY Faith WEIL
• 金额: $ 5.15万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8782964
• 关键词: Address; Affect; Amino Acid Sequence; Amino Acids; Attenuated; Binding; Bypass; Code; Codon Nucleotides; Complex; Conflict (Psychology); Data; Disease; Dyes; Elements; Event; Exhibits; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Frequencies; Genome; Label; Lead; Light; Literature; Mechanics; Messenger RNA; Modification; Mutation; Pathway interactions; Peptide Sequence Determination; Phenylalanine-Specific tRNA; Physiology; Process; Protein Biosynthesis; Protein Conformation; Proteins; Purines; Reporter; Reporting; Research; Ribosomal RNA; Ribosomes; Side; Signal Transduction; Silent Mutation; Stochastic Processes; Stretching; Structure; Time; Transfer RNA; Translating; Translations; base; design; human disease; melting; protein folding; protein misfolding; public health relevance; purine; research study; single-molecule FRET

DESCRIPTION (provided by applicant): Ribosomal protein synthesis is a discontinuous process, with pauses modulating the rate of translation. Such pauses can be crucial for the successful completion of co-translational events such as protein folding and side chain modification4. Codon and codon pair usage, internal Shine-Dalgarno sequences and downstream mRNA secondary structures are a few of the known translational pausing elements. However, the magnitude to which these elements induce pausing has not yet been quantified. In this proposal, the pausing elements stated above will be evaluated for their effect on the rate of translation. Because translation elongation is a stochastic process, it is impossibl to synchronize ribosomes for ensemble studies. To bypass this limitation, single molecule fluorescence resonance energy transfer (smFRET) experiments will be performed and visualized by total internal reflection fluorescence (TIRF) microscopy in order to follow the translation rate of single ribosomes. In these experiments, FRET signals are transiently generated when Phe-tRNAPhe (labeled with a FRET acceptor dye) is delivered to the ribosome (labeled with a FRET donor dye) during the translation of EmGFP mRNA. The FRET signals are then assigned to the respective Phe residues within the EmGFP protein sequence to define discrete translated segments, and the translation time of each segment is quantified. Using this platform, pausing elements will be inserted into the segments of EmGFP to determine their effect on translation rate. These studies will shed light on the magnitude to which these elements affect the rhythm of translation elongation. In addition, these studies should also shed light on the potential effects of "silent" synonymous mutations (mutations that change the codon identity but not the amino acid it codes for), which have been associated with protein misfolding and changes in physiology.

描述(申请人提供)：核糖体蛋白质合成是一个不连续的过程，停顿调节翻译的速度。这种停顿对于成功完成共翻译活动至关重要，例如蛋白质折叠和侧链修饰4。密码子和密码子对的使用、Shine-Dalgarno的内部序列和下游的mRNA二级结构是几个已知的翻译暂停元件。然而，这些因素导致停顿的程度尚未量化。在本提案中，将评估上述停顿因素对翻译速度的影响。因为翻译伸长是一个随机过程，所以不可能同步核糖体进行系综研究。为了绕过这一限制，将进行单分子荧光共振能量转移(SmFRET)实验，并通过全内反射荧光(TIRF)显微镜进行可视化，以跟踪单个核糖体的转化率。在这些实验中，当Phe-tRNAPhe(用FRET受体染料标记)在EmGFP mRNA翻译过程中被传递到核糖体(用FRET供体染料标记)时，FRET信号是瞬时产生的。然后将FRET信号分配给EmGFP蛋白序列中的各个Phe残基，以定义离散的翻译片段，并量化每个片段的翻译时间。利用这个平台，在EmGFP的片断中插入停顿元素，以确定它们对翻译速度的影响。这些研究将阐明这些因素在多大程度上影响翻译延长的节奏。此外，这些研究还应该阐明“沉默的”同义突变(改变密码子身份但不改变密码子编码的氨基酸的突变)的潜在影响，这些突变与蛋白质错误折叠和生理变化有关。