Effects of Translational Pausing Elements on the Rhythm of Protein Synthesis

描述（由申请人提供）：核糖体蛋白质合成是一个不连续的过程，暂停调节翻译速率。这种停顿对于成功完成共翻译事件（如蛋白质折叠和侧链修饰）至关重要。密码子和密码子对的使用、内部Shine-Dalgarno序列和下游mRNA二级结构是一些已知的翻译暂停元件。然而，这些因素引起暂停的程度尚未量化。在本提案中，将评估上述暂停因素对翻译速度的影响。由于翻译延伸是一个随机过程，因此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）显微镜观察，以跟踪单个核糖体的翻译速率。在这些实验中，当在EmGFP mRNA的翻译期间将Phe-tRNAPhe（用FRET受体染料标记）递送至核糖体（用FRET供体染料标记）时，瞬时产生FRET信号。然后将FRET信号分配给EmGFP蛋白质序列内的相应Phe残基，以定义离散的翻译片段，并定量每个片段的翻译时间。使用该平台，将暂停元件插入EmGFP片段中，以确定其对翻译速率的影响。这些研究将揭示这些因素对翻译延伸节奏的影响程度。此外，这些研究还应该阐明“沉默”同义突变（改变密码子身份但不改变其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。

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.