Structure and function of pseudouridine synthases

Pseudouridine (the 5-ribosyl isomer of uridine) is the single most abundant post-transcriptional modification found in the stable, structured RNAs (ribosomal, transfer, small nuclear, and small nucleolar) of all organisms. Despite the fact that it was discovered circa 55 years ago, the function of pseudouridine remains mysterious. Although the lack of pseudouridine in RNAs contributes to translational and splicing defects and is implicated in one rare human disease, dyskeratosis congenita, the mechanisms remain obscure. Pseudouridines are synthesized from uridines at specific locations in RNA by enzymes called pseudouridine synthases. All known pseudouridine synthases can be grouped into five distinct families (TruA, TruB, TruD, RsuA, RluA) according to sequence homology, and each is named for the first Escherichia coli member to be characterized biochemically.To date, E. coli is the only organism in which the complete set of rRNA and tRNA pseudouridines and responsible pseudouridine synthases have been determined, and, therefore, it is a model system to investigate the function of pseudouridine and pseudouridine synthases. In this work, I identified two of these E. coli pseudouridine synthases RluB and RluE as responsible for pseudouridines 2605 and 2457, respectively, in 23S rRNA. I determined the crystal structures of the catalytic domains of the E. coli RluA family rRNA-specific pseudouridine synthases RluD and RluC to try to understand the nature of their multisite specificity. I also participated in the solution of the structure of the most recently discovered E. coli tRNA-specific pseudouridine synthase TruD. I found that all five families of pseudouridine synthases share a catalytic domain fold, active site placement, active site architecture, and sequence/structural motifs, and they likely arose by divergent evolution. In addition, I participated in the location of four pseudouridines (analogous to E. coli 1911, 1915, 1917 and 2605) in the entire 23S rRNA of Deinococcus radiodurans which allowed us to examine the three-dimensional environment of pseudouridine in the D. radiodurans 50S structure. I also identified two D. radiodurans pseudouridine synthases responsible for these pseudouridines