Probing Ribosome Assembly

核糖体是由三种不同的核糖体RNA和50多种不同的核糖体蛋白组成的大分子复合体。这些络合物可以在体外，在精心定义的条件下组装。在组装过程中，随着蛋白质的加入，核糖体RNA(RRNA)结构会发生复杂的变化，将复合体转变为功能核糖体亚单位。随着每个蛋白质的加入，这一过程中的各个步骤将通过评估rRNA的部分可用来使用硫酸二甲酯进行修饰来进行分析。这将在将某些核糖体蛋白添加到rRNA之后在毫秒内完成。这项名为快速非平衡探测的新技术将为rRNA中发生的瞬时结构变化提供重要的洞察力，这些变化是发展功能性核糖体所必需的。这种洞察对于理解蛋白质/RNA的相互作用至关重要，因为很明显，结合蛋白质的过程涉及rRNA的结构变化。此外，快速非平衡探测技术的进一步发展有望在许多领域产生广泛影响，因为它可以应用于几乎任何系统的功能，包括核酸-蛋白质相互作用和蛋白质-蛋白质相互作用，只要修饰反应时间尺度合适。此外，随着这一提议中描述的功能强大的软件的继续开发，将有可能开发这种相互作用的原子细节的动力学模型。该项目的一个更广泛的影响是，学生将能够参与研究蛋白质/核糖核酸相互作用的一种新方法的开发和扩展，其中将涉及核糖体、核糖体蛋白质、核糖体核糖体核糖核酸和引物延伸测序技术的“动手”制备。此外，还将有很好的机会开发更多的计算机建模软件，对核糖体组装时发生的结构变化进行建模。

Ribosomes are macromolecular complexes composed of three different ribosomal RNAs and over 50 different ribosomal proteins. These complexes can be assembled, in vitro, under carefully defined conditions. During the assembly process, complex changes in the ribosomal RNA (rRNA) structure occur as proteins are added, transforming the complexes into functional ribosomal subunits. The individual steps in this process, as each protein is added, will be analyzed by assessing the availability of portions of the rRNA to modification using dimethyl sulfate. This will be done in millisecond time frames following the addition of certain ribosomal proteins to the rRNA. This novel technique, termed rapid non-equilibrium probing, will provide significant insight into transient structural changes occurring in the rRNA that are necessary to develop functional ribosomes. Such insight is critical to understanding protein/RNA interactions, since it is clear that the process of binding the proteins involves structural changes in the rRNA. In addition, further development of the rapid non-equilibrium probing technique promises widespread impact on many fields, since it can be applied to virtually any system as it functions, including nucleic acid-protein interactions and protein-protein interactions, providing the modification reaction time scales are suitable. Additionally, with the continued development of the powerful software described in this proposal, it will be possible to develop kinetic models of such interactions in atomic detail. A broader impact of this project is that students will be able to participate in the development and expansion of a novel approach in the study of protein/RNA interactions which will involve "hands-on" preparation of ribosomes, ribosomal proteins, ribosomal RNA and primer extension sequencing techniques. In addition, there will be excellent opportunities to develop additional computer modeling software to model the structural changes that occur as the ribosome is assembled.