Rapid Probing of EF-G Interactions with Ribosomes

描述(申请人提供)：核糖体是一种复杂的分子机器，它将遗传信息处理成生命所必需的蛋白质产品。核糖体功能的核心是能够准确地选择携带适当氨基酸的tRNAs，并将它们与mRNA中的密码子信息一起在准确的框架内移动(移位)。这一易位过程需要大小核糖体亚基以一种严格的方式进行沟通，以将信息和不断增长的新生肽链转移到一个富有成效的终点。我们建议利用快速非平衡探测(RNP)结合其他生化方法来研究EF-G对这一过程的影响，以确定当EF-G在易位过程中被添加和结构修改时，rRNA中发生的随时间的变化。所提出的模型是EF-G作为俘获能力，使tRNAs的运动与mRNAs的运动同步。通过分析16S和23S rRNA特定区域发生的溶剂可及性变化，我们将识别易位过程中与时间相关的事件。这一建议是高风险的，因为没有关于EF-G在核糖体上的活性的详细的动力学研究。然而，影响是巨大的，因为如果这一假设被证明是正确的，它将使我们能够理解这种同步运动是如何在核糖体上发生的，这反过来将为其他系统中类似的协调过程提供洞察。

DESCRIPTION (provided by applicant): Ribosomes are complex molecular machines that process genetic information into protein products essential for life. At the heart of ribosome function is the ability to accurately select tRNAs carrying appropriate amino acids and move (translocate) them in exact frame with the codon messages occurring in mRNA. This translocation process demands that the large and small ribosomal subunits communicate in an exacting way to move the message and growing nascent peptide chain to a fruitful endpoint. We propose to study the effect of EF-G on this process, using rapid non-equilibrium probing (RNP), in conjunction with other biochemical approaches, to identify the time-dependent alterations occurring in rRNA as EF-G is added and is structurally modified in the translocation process. The model proposed is that EF-G acts in an escapement capacity, synchronizing the movement of the tRNAs with that of mRNA. By analyzing changes in solvent accessibility that occur in specific regions of 16S and 23S rRNA, we will be to identify the time- correlated events of the translocation process. This proposal is high risk since there are no detailed kinetic studies on the activities of EF-G on the ribosome. However, the impact is great, since if this scenario proves to be correct, it will allow us to understand how such synchronous movements take place on the ribosome, which in turn will provide insight on similar coordinated processes in other systems.