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Pseudouridine synthases are enzymes responsible for modifying uridines to pseudouridines in a site-specific and energy-independent manner. There are 5 families of these synthases, named after the first member of each family to be characterized in Escherichia coli : RluA, RsuA, TruA, TruB and TruD. The 23S ribosomal RNA in E. coli contains 10 pseudouridine modifications made by 6 specific synthases named RluA-RluF. These modifications cluster around important functional regions of the ribosome such as the peptidyl transferase center, the tRNA binding sites, and the inter-subunit bridge regions. My research focuses on understanding the mechanisms of substrate selection by pseudouridine synthases and the roles of these modifications in ribosome biogenesis and function. The main aims of my research were: a) to examine the substrate specificity determinants of RluD, an important E. coli synthase; b) to characterize a mutant strain of E. coli lacking a majority of the pseudouridines on 23S rRNA; and c) to determine the activity of RluA from Vibrio cholerae, which has two closely related RluA paralogs rather than just one, as seen in most organisms. Pseudouridine modifications in the stem-loop of helix 69 (H69) in domain IV of 23S ribosomal RNA are highly conserved in all phyla. The three pseudouridines in H69 in E. coli have been shown to play an important role in 50S subunit assembly and its association with the 30S subunit. These three modifications are made by the pseudouridine synthase, RluD. Previous work showed that RluD is required for normal ribosomal assembly and function, and is the only pseudouridine synthase required for normal growth in E. coli. Here, we show that RluD is far more efficient in modifying H69 in structured 50S subunits, rather than in naked or synthetic 23S rRNA. We suggest that pseudouridine modifications in H69 are made late in the assembly of 23 rRNA into the mature 50S subunit. This is the first reported observation of a pseudouridine synthase being able to modify a structured ribonucleoprotein particle, and may constitute an important late step in the maturation of 50S ribosomal subunits. Deletion of RluD results in aberrant ribosome assembly and impaired translation termination leading to severe growth defects. However, single deletion strains of the remaining five 23S rRNA synthases do not display an altered phenotype. In an effort to identify possible roles for the remaining seven pseudouridines, we constructed a strain (Delta 5 mutant) lacking all 23S rRNA synthases except RluD. Surprisingly, this strain does not exhibit a significant growth defect at 37C in rich or minimal media. However, it does display a slower growth rate at 20C compared to wild-type. When grown in competition with the wild-type strain at 37C, a strong selection against the mutant strain was observed. In order to evaluate the structure of the mutant ribosomes, we determined the effect of various antibiotics that target the 50S subunit. The mutant strain is significantly more sensitive than wild-type to antibiotics targeting the 50S subunit such as chloramphenicol, hygromycin, clindamycin and tiamulin but these effects can be attributed to the loss of the RluC modification at U2504 by itself. In phenotypic microarray tests, we observed that the Delta 5 mutant grew much poorer than wild-type when cultured in a medium containing 6% NaCl. Taken together, the data suggest that these pseudouridines may play an important role in maintaining the structural integrity of the ribosome. In E. coli, the pseudouridine synthase RluA is a dual specificity synthase capable of modifying U746 on 23S rRNA and U32 on 4 cytoplasmic tRNAs. Surprisingly, the Vibrio cholerae genome encodes not one, but two closely related RluA proteins. In order to examine the possible activities of these two proteins, we complemented an rluA deletion in E. coli with plasmid-borne Vibrio rluA1 and rluA2 constructs. Interestingly, only one of these two RluA proteins (Vibrio RluA1) was able to modify E. coli 23S rRNA at U746. In order to determine the structural basis for this difference between the closely related RluA1 and RluA2, we constructed homology models using the structure of E. coli RluA in complex with an RNA stem-loop (PDB ID: 2I82) as a template. These models implicated two possible three amino acid (GVF or FAL) inserts present near the catalytic aspartate in Vibrio RluA2 as the likely cause of the differential activity. We hypothesize that this insert may sterically occlude the binding of substrate RNA to the enzyme, thereby preventing a productive modification reaction.

Pseudouridine is the most common RNA modification found in all forms of life. The exact role pseudouridines play in the cell is still relatively unknown. However, its extensive incorporation in functionally important areas of the ribosome and the fitness advantage provided to cells by pseudouridines implies that its presence is important for the cell. The enzymes responsible for this modification, pseudouridine synthases, vary greatly in substrate recognition mechanisms, but all enzymes supposedly share a universally conserved catalytic mechanism. Here, I analyze the kinetic mechanisms of pseudouridylation utilized by the exemplary pseudouridine synthase RluA in order to compare it with the previously determined rate of pseudouridylation of the pseudouridine synthase TruB. My results demonstrate that RluA has the same uniformly slow catalytic step as previously determined for TruB and TruA. This constitutes the first step towards identifying the catalytic mechanism of the pseudouridine synthase family. Additionally, it was my aim to identify the major determinants for RNA binding by pseudouridine synthases. By measuring the dissociation constants (KD) for substrate and product tRNA by nitrocellulose filtration assays, I showed that both tRNA species could bind with similar affinities. These binding studies also revealed that TruB’s interaction with the isolated T-arm is the major contact site contributing to the affinity of the enzyme to RNA. Finally, a new contact between tRNA and TruB’s PUA domain was identified which was not observed in the crystal structure. In summary, my results provide new insight into the common catalytic step of pseudouridine synthases and the specific interactions contributing to substrate binding by the enzyme TruB. These results will enable future studies on the kinetic mechanism of pseudouridine synthases, in particular the kinetics of substrate and product binding and release, as well as on the chemical mechanism of pseudouridine formation. / xi, 122 leaves : ill. (some col.) ; 29 cm

Over one hundred types of chemical modifications have been characterized in cellular RNAs. Pseudouridines (Ψ) and 2’-O-methylation of ribose sugars are the two most widespread modifications present in rRNAs, tRNAs and snRNAs. These modifications can be either guide-RNA mediated or RNA-independent (enzyme only). The RNAs that guide pseudouridylations are called box H/ACA RNAs and the ones that carry out 2’-O-methylations modifications are called box C/D RNAs. Previously, we identified that sR-h45 is the box H/ACA guide RNA responsible for Ψ1940, 1942 and 2605 formation in 23S rRNA of Haloferax volcanii. This RNA has two stem loops – SL1 and SL2. SL1 acts as the guide for Ψ2605 formation and SL2 is responsible for guiding Ψ1940 and Ψ1942. We found that SL2 sequentially guides Ψ1940 and Ψ1942 formation in the unpaired "UNUN" target. Ψ1942 is produced after and only if Ψ1940 is produced. The requirement for conserved ACA box was determined by using variants of these two stem loops. We found that the ACA motif is not required either in vivo or in vitro for the activity of the typical variants of both SL1 and SL2 but required for the activity of the atypical variants of these guides. Cbf5 is the pseudouridine synthase involved in this box H/ACA RNA guided process. Mutants of Methanocaldococcus jannaschii Cbf5 were used with both typical and atypical guide variants in vitro and certain residues were found to be important only for the atypical reactions.We have also studied sR-h41, which is a unique single guide box C/D guide responsible for methylation of G1934 position of 23S rRNA of Haloferax volcanii. We have done in vitro assembly reactions using mutants of sR-h41 assembled with its cognate proteins from Methanocaldococcus jannaschii to study the structural determinants needed to convert it to a dual guide RNA. The assembly pattern of the core proteins on the conserved box C/D and box C’/D motifs steer the dual guide nature of these archaeal box C/D guide RNAs.Another aim of this study was to determine the role of pseudouridine synthases (Pus enzymes) for Ψ55 formation in mammalian tRNAs. We find that three Pus enzymes – TruB1 (in the nucleus), TruB2 (in the mitochondria) and Pus10 (in the cytoplasm) are responsible for this modification depending on the specific sub-cellular location in the cell. These enzymes exhibit different structural requirement for Ψ55 formation that are located on the TΨC loop of tRNAs. A subset of tRNAs like tRNAs for Trp and Gln are protected from the action of TRUB1 in the nucleus by binding to the nuclear version of Pus10 that lacks Ψ55 activity. Ψ55 in this subset of tRNAs is produced by the cytoplasmic version of Pus10.While studying pseudouridylation functions of Pus10, we also found that Pus10 regulates G1/S cell cycle progression in PC3 cells. It does so by directly repressing another protein c-Rel, that is a positive regulator of Cyclin D1 protein. Cyclin D1 is known to play a central role in transition of cell from G1 to S phase during cell cycle progression. c-Rel also regulates the levels of PUS10 by an unknown mechanism.

No description available.

tRNA molecules contain more than 80 chemically unique nucleotide base modifications that contribute to the chemical and physical diversity of RNAs as well as add to the overall fitness of the cell. For instance, base modifications have been shown to play a critical role in tRNA molecules by improving the fidelity and efficiency of translation. Most of this work has been carried out extensively in Gram-negative bacteria, however, the role of modified bases in tRNAs as they relate to thermostability, structure, and transcriptional regulation in Gram-positive bacteria, such as Bacillus subtilis and Bacillus anthracis, are not well characterized. Infections by Gram-positive bacteria that have become more resistant to established drug regiments are on the rise, making Gram-positive bacteria a serious threat to public safety. My thesis work examined what role partial base modification of the tyrosyl-anticodon stem-loops (ASLTyr ) of B. subtilis and B. anthracis have on thermostability, structure, and transcriptional regulation. The ASLTyr molecules have three modified residues which include Queuine (Q34), 2-thiomethyl-N6-dimethylallyl (ms2i6A37), and pseudouridine (Y39). Differential Scanning Calorimetry (DSC) and UV melting were employed to examine the thermodynamic effects of partial modification on ASLTyr stability. The DSC and UV data indicated that the Y39 and i6A37 modifications improved the molecular stability of the ASL. To examine the effects of partial base modification on ASLTyr structure, NMR spectroscopy was employed. The NMR data indicated that the unmodified and [Y39]-ASLTyr form a protonated C-A+ Watson-Crick-like base pair instead of the canonical bifurcated C-A+ interaction. Additionally, the loop regions of the unmodified and [Y39]-ASLTyr molecules were well ordered. Interestingly, the [i6A37]- and [i6A37; Y39]- ASLTyr molecules did not form a protonated C-A+ base pair and the bases of the loop region were not well ordered. The NMR data also suggested that the unmodified and partially modified molecules do not adopt the canonical U-turn structure. The structures of the unmodified, [Y39]-, and [i6A37;Y39]-ASLTyr molecules did not depend on the presence of Mg2+, but the structure of the [i6A37]-ASLTyr molecule did depend on the presence of multivalent cations. Finally, to determine the repercussions that partial modification has on physiology and tRNA mediated transcriptional regulation in B. anthracis, antibiotic sensitivity tests, growth curves, and quantitative real-time polymerase chain reaction (qRT-PCR) were employed. Strains deficient in ms2 showed comparable growth to the parent strain when cultured in defined media, but Q deficient strains did not. The loss of ms2i6A37 conferred resistance to spectinomycin and ciprofloxacin, whereas the loss of Q34 resulted in sensitivity to erythromycin. Changes in the ratio full-length to truncated transcripts of the tyrS1 and tyrS2 genes were used to monitor tRNA mediated transcriptional regulation. The qRT-PCR data suggested that tyrS1 and tyrS2 are T-box regulated and that the loss of ms2i6A37 and Q34 might affect the interaction of the tRNATyr molecule with the specifier sequence, which is located in the 5’-untranscribed region (UTR) of the messenger RNA (mRNA).

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京都大学 / 新制・課程博士 / 博士(理学) / 甲第23724号 / 理博第4814号 / 新制||理||1689(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM