The objective is to elucidate the structure-function relationship of the eukaryotic ribosome. The overall aim is to exploit recent advances indicating that functional characteristics of the ribosome is closely related to conformational changes in, and dynamic properties of its complex structure. We will focus on the dynamics of the yeast ribosome and its subunits.
Specific aims are to: 1. continue our studies in defining the contribution of individual proteins to specific ribosomal functions by partial reconstitution in vitro, 2. selectively label ribosomal proteins with fluorescent probes under non-denaturing conditions and reconstitute biologically active ribosomal subunits with fluorescent-labeled protein(s), focusing on those proteins whose functions have been demonstrated, 3. characterize the properties of ribosomal subunits containing a specific fluorescent-labeled protein by both steady-state and nanosecond fluorescence, 4. study variations in fluorescence properties as the fluorescent ribosome interacts with other components of the protein synthetic machinery, 5. determine the kinetics of tritium exchange of yeast ribosomal subunits that are in different functional states and attempt to identify individual proteins whose dynamic properties are changed as the ribosome is engaged in different biological activities using low pH HPLC, and 6. identify protein neighborhood of selective ribosomal proteins, focusing on the """"""""acidic"""""""", phosphoproteins and ribosomal subunit interface proteins. The dynamics of the ribosome will be studied using techniques that have been applied successfully to monitor dynamics of proteins, viz. fluorescence and tritium exchange to define global and local changes in ribosomal proteins. These changes will be correlated with specific functional states in which the ribosome is engaged during protein synthesis. Protein topography will be determined by chemical crosslinking using several reversible bifunctional reagents. Crosslinked products will be identified by 2-D polyacrylamide gel electrophoresis, HPLC and immunoblotting. A noval approach, using radiolabeled heterobifunctional asymmetric and cleavable reagent, will be used to identify subunit interfacial proteins. Information derived from the fluorescence and tritium exchange studies will be correlated with functional as well as static information of the ribosome.
