The long-term objective of the research is to elucidate the structure-function of the yeast ribosome. The present proposal focuses on the 5S RNP ribosomal domain containing the 5S RNA and its binding protein Lla in the 60S subunit. The structure of the 5S RNP, the """"""""acidic"""""""" proteins and proteins L7 and L26 are affected during subunit association. Our working hypothesis is that these components are located at or near the 6OS-40S interace and that modulation of the interface can regulate ribosome activity.
Specific aims are to: (1) identify proteins in the vicinity of protein Lla in the intact ribosome by protein-protein crosslinking; (2) determine the location of the N- and C-termini of protein Lla in the ribosome by using site-directed mutagenesis to introduce specific cysteine residues and subsequent disulfide crosslinking; (3) use immunoelectron microscopy to map Lla in the intact ribosome with respect to the """"""""acidic"""""""" proteins, proteins L7 and L26; (4) identify proteins that are near proteins L7 and L26 by protein-protein crosslinking and their relation to the 5S RNP; (5) use site-directed mutagenesis to generate protein Lla mutated at the 6 potentially critical lysine residues in the C-terminal region; (6) determine how the modified protein Lla interacts with 5S RNA, reconstitutes into 60S subunit, and participates in protein synthesis; (7) generate 5S RNA species mutated in the region between residues 20-45; and (8) determine how the modified 5S RNA molecule interacts with protein Lla, reconstitutes into 60S, and participates in ribosomal subunit interaction. These data together with the results of the correlated functional studies will be used to construct a model depicting the spatial arrangement of proteins in or near the 5S RNP domain in the yeast ribosome. Knowledge about the 5S RNP in the yeast ribosome is important because the 5S RNP is essential for ribosomal activity and also because the 5S RNP is an early assembly intermediate. This information is essential for understanding the mechanism of action of the ribosome, an integral part of all living cells.
