Kinetic Studies of the 30S Ribosomal Subunit Assembly Mechanism

描述(由申请人提供)：本提案概述了一项研究30S核糖体亚单位组装机制的计划，特别是确定体外组装的途径和测量共转录组装的效果。我们的目的是通过(1)测量单个核糖体蛋白的结合率并用三色双光子激发荧光检测组装过程中填充的中间产物，以及(2)设计一种用于30S组装的体外共转录实验，来回答有关组装过程中的协同程度和限速步骤(S)的性质的问题。方法：为了获得可能中间体的动力学信息，我们正在建造一台双光子激发三色检测荧光显微镜，使我们能够测量单分子到小集合水平的动力学速度。为了实时测量通量，我们将使用依赖时间的荧光相关光谱(PCS)一次研究多达三种蛋白质的关系，每种蛋白质都标记了不同的荧光标签。我们将测量相对速率常数，并通过监测特定蛋白质的速率在另一种蛋白质存在时是否发生变化来寻找动力学协同性。通过自动关联荧光强度信号，我们将确定每个物种在时间上的浓度和单个结合率，并使用互相关测量来量化高阶分子络合物的形成。相关：一个功能正常的核糖体对细胞存活至关重要，了解组装过程是如何调节的在生物学上具有多种含义。这些研究将有助于定量定义30秒的能量格局，并通过识别1)多条途径是否能导致完整的活性核糖体的形成，以及这些途径是什么，2)任何中间产物的组成，3)共转录组装对组装过程速度的影响，从而对组装过程产生机制的洞察。

DESCRIPTION (provided by applicant): This proposal outlines a plan to investigate the assembly mechanism of the 30S ribosomal subunit, specifically to determine the pathways of assembly in vitro and to measure the effect of co-transcriptional assembly. Our aim is to answer questions about the extent of cooperativity in the assembly process and the nature of the rate-limiting step(s) by (1) measuring binding rates of individual ribosomal proteins and detecting any intermediates that are populated during assembly by three-color, two-photon excitation fluorescence and (2) designing an in vitro co-transcriptional assay for 30S assembly. Method: In order to obtain kinetic information on possible intermediates, we are constructing a two-photon excitation three-color detection fluorescence microscope that allows us to measure kinetic rates at the single molecule to small ensemble level. To measure the fluxes in real-time we will use time dependent fluorescence correlation spectroscopy (PCS)to study the relationships of up to three proteins at a time, each labeled with a different fluorescent tag. We will measure the relative rate constants and look for kinetic cooperativity by monitoring if the rates of a particular protein change in the presence of another protein. By auto-correlating the fluorescence intensity signal we will determine the concentrations of each species in time and individual binding rates and quantify higher order molecular complex formation using cross-correlation measurements. Relevence: A properly functioning ribosome is crucial to cell viability and understanding how the assembly process is regulated has a multitude of implications in biology. These studies will help to quantitatively define the energy landscape of 30S and yield mechanistic insights into the assembly process by identifying 1) if multiple pathways can lead to the formation of the intact active ribosome and what these pathways are 2) the composition of any intermediates and 3) the effect of co-transcriptional assembly on the rate of the assembly process.