Single Molecule Dynamics of mRNA Translation

翻译速率的调节对于这种下游共翻译前体至关重要。 如蛋白质折叠和修饰、配体结合、寡聚化以及与 分子伴侣和膜。虽然调节翻译速率的特定元素很好地 众所周知，目前几乎完全缺乏描述这些元素如何 影响翻译速度。我们的总体目标是通过量化 E.特异性mRNA和新生肽的大肠杆菌核糖体 序列，并阐明这种调制的机制。我们使用一种方法， 全内反射荧光显微镜（TIRFM），单分子荧光 共振能量转移（smFRET）。引入荧光报告子和smFRET对 特别是氨酰转移RNA和核糖体蛋白L1和L11，它们是近端的 分别连接到tRNA的E位点和A位点。这些荧光标记的化合物的可用性， 蛋白质合成机制的基本原理允许在单个核糖体上观察， FRET相互作用的时间监测氨酰-tRNA结合到核糖体A-位点，interme- tRNA在相邻位点的径向运动，tRNA向P-和E-位点的易位，以及 从E位点释放释放出的tRNA。单分子测定已经开发， 对于强调与荧光tRNA配对的短模型mRNA和编码 具有所有氨基酸的天然互补的全长蛋白质。一个快速的表达 绿色荧光蛋白的成熟变体Emerald GFP（EmGFP）通过荧光信号传递， TIRF显微镜中单个EmGFP分子的荧光。总之，这些方法允许 在延伸周期内的单个步骤的辨别和连续动力学曲线 mRNA翻译，提供了独特的见解，以调节翻译速率在prokary- 耳蛋白合成不能从其他技术获得。我们的研究结果将使我们能够量化 调节元素的影响，并确定是否新的元素和协同或antago- 免疫效应是有效的。正确解释我们的结果的关键是性能 控制实验表明，我们测量的速率效应来自于引入 的预期的调制元件，而不是从一个或多个其他元件的参与， 的部分。我们有3个具体目标，重点是确定蛋白质合成的节奏如何 通过以下方式调制：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. 暂停元素组合出现。总的来说，我们研究几个暂停电子- 在完整蛋白表达水平上和在单个延伸循环内的片段将定义 翻译调节的大小和详细机制。

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.