In all cells, the process of protein synthesis proceeds on ribosomes, one of the most ancient of all macromolecular structures found in the cell. The functional heart of the ribosome is its RNA, one high molecular weight molecule for each subunit, complexed with a characteristic set of proteins. Ribosomal RNA contains a number of modified nucleosides with the isomer uridine called pseudouridine ( psi) being the most prevalent single modification found. Psi residues are not distributed at random but are found at specific sites, many of which recur from species to species. In prokaryotes, specific enzymes are responsible for the placement of psi at particular sites in ribosomal RNA, while in eukaryotes, an elaborate system of guide RNAs has evolved for placing the psi residues correctly. Despite this careful placement of psi at specific sites in the ribosome, the role of psi in ribosome structure nad/or function is unknown. The specific objective of this proposal is an understanding of how psi residues are made in the ribosome and what their role is in its overall workings, using Escherichia coli as the model system. The specific aims are: (1) to determine the role played by psi at particular sites in ribosomal RNA by deleting them one by one and analyzing the effect on the cell's ability to make protein. Specific deletion will be accomplished by disruption of the gene for the specific enzymes responsible for the synthesis. (2) to understand the RNA-protein recognition mechanisms which these enzymes use to pick out one U out of over 500 others. (3) to determine the stage of ribosome formation at which the psi residues are made. (4) to search for possible progenitors of the eukaryotic guide RNAs in E. coli postulating that before they acquired their site selection function they may have functioned as reaction rate enhancers. The long-term goal is to elucidate the mechanisms of ribosomal RNA function in ribosome at the most basic molecular level. As the process of protein synthesis is so fundamental to all cells, information of this kind cannot help but advance our knowledge of the physiology of the cell both in healthy and diseased states.