Probing Ribosome Assembly

University Of Montana, Missoula MT

Investigators

Abstract

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.