Pseudouridine (psi), the most common modified nucleoside in RNA, occurs in ribosomal RNA (rRNA) ( transfer RNA (tRNA), and small nuclear (sn) and nucleolar (sno) RNAs, but not in messenger RNA. Psi is made by enzyme-catalyzed isomerization of specific uridines in a pre-formed polynucleotide. This unique reaction involves breaking the uridine N-C glycosyl bond, rotating the uracil base, and reforming a C-C glycosyl link. Two classes of enzyme systems exist. In eubacteria, all psi are made by a set of site-specific proteins. In archaea and eukarya, psi in tRNA and some sn(o) RNAs are made by specific proteins also, but psi in rRNA and other sn(o)RNAs are made by a ribonucleoprotein complex containing a guide RNA for site selection. Genes for psi synthases occur in the most ancient genomes, indicating that ( was an old invention that has persisted for millennia. In this work, the role of psi and psi synthases will be studied using E, coli as the model organism. E. coli has 11 psi sites in rRNA and 7 in tRNA. The psi are made by 11 different synthases because some make multiple psi. The only synthase deletion which markedly inhibited growth was that of RluD, a synthase responsible for 3 psi found at the same place in almost all 50S ribosomes. 50S ribosomes from this strain were highly abnormal, and appeared blocked at some stage of assembly. We plan to (1) study the mechanism by which deletion of RluD blocks ribosome assembly, (2) determine the X-ray structure of RluD-substrate complexes in order to understand the nature of this reaction, (3) study the features of the RNA substrate that allow specific recognition by RluD, and (4) look for physical and/or functional partners of RluD and the other synthases by a synthetic lethal screen and by searching for association in the cell by """"""""pull-down"""""""" experiments. This work will contribute to our understanding of the basic biology of the cell. Moreover, since RluD is so necessary for growth and appears absent in eukaryotes, potential exists for the development of antibacterial therapies.
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