Identification of three Escherichia coli pseudouridine synthases, and characterization of TruD structure and function

Pseudouridine (5-ribosyluracil; Psi) is the most common modified nucleoside found in structural RNAs (ribosomal, transfer, small nuclear, and small nucleolar RNAs). Over 50 years after its discovery as a fifth nucleoside, a unitary function for all Psis remains elusive. Pseudouridines are synthesized at the polynucleotide level by the enzymes called pseudouridine synthases. Pseudouridine synthase genes are members of a class consisting of five subgroups that share a common catalytic domain architecture and active site organization. The five families of pseudouridine synthases (RsuA, RluA, TruA, TruB, and TruD) are named after the first Escherichia coli member of each family to be identified experimentally.In our laboratory, we work on structural and functional characterization of Psi and Psi synthases using Escherichia coli as a model organism. In this work, I identified the sites of action for two putative pseudouridine synthases, RluF and TruC, which were found to act on Psi2604 in 23S rRNA, and Psi65 in tRNAAsp and tRNAlle1, respectively. I also brought a novel and unanticipated family of pseudouridine synthases, the TruD family, to light using standard biochemical procedures. Bioinformatic analysis showed that the TruD family is widespread, with homologs spanning all three phylogenetic domains, including ancient organisms. I characterized six motifs shared among the TruD family that have no sequence homology, except for a conserved aspartate residue in motif II, to the four previously known Psi synthase families. I showed that this aspartate is essential for catalytic activity. I solved the crystal structure of TruD to 2.2 A° resolution, and showed that all five families of pseudouridine synthases possess a common catalytic domain. Finally, I also determined kinetic parameters for wild-type TruD and two Asp 80 mutants, D80N and D80T.TruD is responsible for modifying uridine13 in tRNAGlu to pseudouridine. Phylogenetic analysis and comparison of the frequency of synonymous and nonsynonymous substitutions among TruD homologs indicate that the TruD enzyme and/or the product pseudouridine13 are important to the cell and under positive selection. I examined the effect of the absence of TruD and Psi13 on tRNA stability and cell growth, but no substantial effects were seen. At elevated temperatures, the absence of TruD confers a slight growth defect in E. coli. Surprisingly, this appears to be independent of the failure to synthesize Psi13, as catalytically inactive TruD mutants do not exhibit a similar growth defect