Kinetic Studies of the 30S Ribosomal Subunit Assembly Mechanism

描述（由申请人提供）：本提案概述了研究30S核糖体亚基组装机制的计划，特别是确定体外组装途径和测量共转录组装的影响。我们的目的是回答有关组装过程中协同性的程度和限速步骤的性质的问题，通过（1）测量单个核糖体蛋白的结合率，并通过三色双光子激发荧光检测组装过程中填充的任何中间体，以及（2）设计用于30S组装的体外共转录测定。方法：为了获得可能的中间体的动力学信息，我们正在构建一个双光子激发三色检测荧光显微镜，使我们能够测量动力学速率在单分子小合奏水平。为了实时测量通量，我们将使用时间相关荧光相关光谱（PCS）来研究一次最多三种蛋白质的关系，每种蛋白质都标记有不同的荧光标签。我们将测量相对速率常数，并通过监测一种特定蛋白质在另一种蛋白质存在下的速率变化来寻找动力学协同性。通过自相关的荧光强度信号，我们将确定每种物质的浓度在时间和个人的结合率和量化更高阶的分子复合物的形成使用互相关测量。发布：一个功能正常的核糖体对细胞的生存能力至关重要，了解组装过程是如何调节的，在生物学上有着广泛的意义。这些研究将有助于定量定义30S的能量景观，并通过确定1）是否有多个途径可以导致完整活性核糖体的形成以及这些途径是什么，2）任何中间体的组成和3）共转录组装对组装过程速率的影响，对组装过程产生机理性见解。

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.