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Although tRNA modifications 5-carboxymethylaminomethyl(-2-thio)uridine (cmnm5(s2)U; and their human homologs) have been linked to mitochondrial disorders, their effects in vivo remain unknown. Furthermore, due to a lack of structural evidence, controversy exists in the literature pertaining to the assembly and catalytic mechanism of the MnmEG complex that introduces these modifications in tRNA. This thesis used a multidisciplinary approach to study the MnmEG modification complex and the effects of cmnm5(s2)U on tRNA structure. Biochemical studies reveal the binding propensities of MnmE and MnmG to their tRNA substrate, contributing vital experimental evidence towards the isolation of the MnmEG-tRNA complex. Concurrently, computational studies uncover the structural and cooperative properties of cmnm5(s2)U in biologically relevant tRNAs, providing the first atomic-level details of the function of these modifications in translation. Overall, this thesis lays the foundation for further explorations into this tRNA modification family and its relation to disease.

Selenocysteine-containing proteins, selenoproteins, are generally favoured over cysteine-containing proteins in redox reactions and therefore of high interest due to their multiple bio-industrial applications. However, the unique features of Selenocysteine biosynthesis and insertion machinery limits the freedom of selenoprotein bioengineering. Whereas engineered Selenocysteine machinery alternatives have been proposed, the role of tRNA post-transcriptional modifications on selenoprotein biosynthesis remains unexplored. This thesis provides insights into the overall positive role tRNA modification on stop codon re-assignment. I have shown that by supplementing coupled transcription-translation cell-free systems with tRNA modifying enzymes, the fluorescence signal of a reporter protein is enhanced compared to protein synthesis in the absence of the modifying enzymes. I conclude that tRNA modifications, in particular N6-isopenthenyladenosine (i6A37), can potentially improve selenoprotein biosynthesis in vitro. Thus, the result of this thesis provides valuable knowledge that could contribute to future optimization of cell-free platforms for selenoprotein bioengineering.

The H/ACA snoRNP complex directs the formation of pseudouridines in ribosomal RNA (rRNA); however, the mechanisms by which the eukaryotic two-hairpin structured H/ACA snoRNP mediates pseudouridylation in rRNA are poorly understood. This study investigates the function of the conserved two-hairpin structure of eukaryotic H/ACA snoRNA while modifying large, structured rRNA. The biochemical analysis reveals that the two hairpins of the snoRNP complex can independently convert two uridines to two pseudouridines in long rRNA fragments. Moreover, the H/ACA snoRNP can likely unfold secondary structures in rRNA without the help of additional factors. Further, the GAR domains of Gar1 are required for pseudouridine formation, but the KKE/D extensions of Cbf5 play no role in pseudouridylation of rRNA. In summary, this study addresses the mechanism of the H/ACA snoRNP complex in modifying rRNA during ribosome biogenesis.

Eukaryotic ribosome assembly is an essential process involving many protein and RNA assembly factors. Mutations of genes encoding ribosomal proteins or assembly factors can result in diseases in humans. The upregulation of ribosome assembly is also a hallmark of many cancers. snR30 is an interesting assembly factor due to its role as an unusual and essential H/ACA small nucleolar RNA and as it is one of only three essential RNA assembly factors. Here, I have developed a system for the co-expression of aptamer-tagged variants of snR30 and truncations of ribosomal RNA in Saccharomyces cerevisiae. Next, I optimized the tandem purifications of the snR30-bound pre-ribosomes utilizing these aptamers. In conclusion, my system will allow for in-depth characterization of the composition and structure of snR30-bound ribosome precursors. This characterization will improve the understanding of the essential activity of snR30 for ribosome assembly in yeast and inform future studies on the function of the homologous U17 RNA in humans.

The creation of eukaryotic ribosomes is a complex process involving hundreds of assembly factors, including the small nucleolar RNA snR30. Through interacting with the unprocessed precursor ribosomal RNA (pre-rRNA), snR30 is essential for the maturation of pre-rRNA forming the small ribosomal subunit. To characterize snR30’s interaction network, an in vitro approach was used to quantify the affinity of snR30 to the core H/ACA protein complex and the PIN endonuclease Utp23 revealing very tight interactions with affinities in the sub- and low-nanomolar ranges, respectively. Furthermore, the snR30 complex binds tightly to its primary binding site in ribosomal RNA called the C1 site, but only weakly to other predicted interaction sites. Utp23 binds tightly to its predicted interaction site in ribosomal RNA only if adjacent rRNA elements are also present. In conclusion, these findings serve as foundational work to further explore the mechanisms of snR30 during maturation of rRNA.

Pseudouridine synthases are RNA modification enzymes targeting various forms of RNA in the cell. RluB and RluF are pseudouridine synthases modifying adjacent uridines in E. coli ribosomal RNA. These enzymes have a highly conserved RNA-binding loop, distinct between RluB and RluF, proposed to play a role in their specificity. This thesis provides preliminary insight into the importance of the eight-residue binding loops of RluB and RluF for RNA target recognition. I have shown that altering the residues of the binding loop decreases the enzyme’s affinity for RNA and effectively abolish the enzyme’s pseudouridylation activity. I conclude that the binding loops of RluB and RluF are critical for RNA binding and for positioning the target RNA for catalysis. Better understanding the requirements for specificity and target recognition of RluB and RluF will enhance our knowledge on ribosome formation and may aid in engineering new enzymes to pseudouridylate other functional RNAs.

Multi-wavelength analytical ultracentrifugation (MW-AUC) is a recent development made possible by new analytical ultracentrifuge optical systems. MW-AUC is suitable for a wide range of applications and biopolymer systems and is poised to become an essential tool to characterize macromolecular interactions. It adds an orthogonal spectral dimension to the traditional hydrodynamic characterization by exploiting unique chromophores in analyte mixtures that may or may not interact. Here we illustrate the utility of MW-AUC for representative classes of challenging biopolymer systems, including interactions between mixtures of different sized proteins with small molecules, mixtures of loaded and empty viral AAV capsids contaminated with free DNA, and mixtures of different proteins, where some have identical hydrodynamic properties, all of which are difficult to resolve with traditional AUC methods. We explain the improvement in resolution and information content obtained by this technique compared to traditional single- or dual-wavelength approaches. We discuss experimental design considerations and limitations of the method, and address the advantages and disadvantages of the two MW optical systems available today, and the differences in data analysis strategies between the two systems.

Pseudouridine is an isomer of uridine and a post-transcriptional RNA modification in all domains of life. Its formation is catalyzed by pseudouridine synthases functioning as standalone proteins or protein-RNA complexes called H/ACA small nucleolar ribonucleoproteins (H/ACA snoRNPs). Whereas pseudouridylation was long known to assist in stabilizing noncoding RNAs, it was discovered also in mRNAs in 2014. mRNA pseudouridylation varies in response to stress, and artificial pseudouridylation of stop codons facilitates nonsense suppression. However, the functional significance of mRNA pseudouridylation and the regulation of the pseudouridine synthases responsible are only partially understood. To study mRNA pseudouridylation by specific pseudouridine synthases, I developed a fluorescence-based reporter system in S. cerevisiae and used it to assay for standalone Pus7, snR5 and snR81 H/ACA snoRNP activity. This novel system detects snR5 H/ACA snoRNP activity and can be used to further investigate the role of pseudouridine synthases in gene expression.

H/ACA guide RNAs are a class of noncoding RNA that direct the pseudouridylation of many cellular RNA species. In most eukaryotes, H/ACA guide RNAs share a conserved hairpin-hinge-hairpin structure, where each hairpin can direct pseudouridylation when associated with evolutionarily conserved core proteins. Target selection occurs by base pairing between target RNA and single-stranded loops within each hairpin of the H/ACA guide RNA, called pseudouridylation pockets. Here, I have analyzed the structure-function relationship of H/ACA guide RNAs by applying a structure-focused approach to design H/ACA guide RNAs for pseudouridylation of novel substrates. Thereby, I designed and tested several artificial H/ACA guide RNAs that were both highly active and specific for their respective substrates in vitro. In addition, I generated multiple sub-optimal H/ACA guide RNA designs that reveal important information regarding H/ACA guide RNA features dictating productivity. My results open new avenues for evaluating, predicting/identifying, and designing cellular guide-substrate RNA combinations.

H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They consist of four proteins and a guide RNA. It is unclear which element of the guide RNA the H/ACA proteins recognize. By designing guide RNA variants with altered structural features, I determined that H/ACA proteins bind guide RNA extremely tightly, but non-specifically in vitro. Additionally, the base-pairing between guide and substrate RNA varies widely in nature. To elucidate substrate selection rules, I systematically altered the sequence of a substrate RNA and tested for snoRNP binding and activity. All substrate RNA variants tested are bound by the H/ACA snoRNP, but not all are effectively modified. Minimal continuous base pairing is required to position the target uridine in the active site for modification, and H/ACA snoRNPs can target structured RNAs. This information will improve the prediction of novel target sites of H/ACA snoRNPs, e.g. in messenger RNAs.

The effect that Dyskeratosis congenita mutations have on ribosome biogenesis has not been fully investigated. Here, I am studying the effect of Dyskeratosis congenita mutations using a yeast model system with the goal of identifying impacts on ribosome biogenesis. I constructed six haploid yeast strains with a single mutation in the CBF5 gene each using site-directed mutagenesis and chromosomal integration. First, I screened for phenotypic effects on growth on solid and in liquid medium at different temperatures; these experiments show normal growth behavior of the mutant strains compared to wild type. Second, Cbf5p expression levels were examined in whole-cell extract via western blotting analysis, which again did not reveal any difference to wild type. Finally, progression through the cell cycle was investigated in synchronized cells. Together, this study indicates that individual Dyskeratosis congenita mutations are well tolerated in the unicellular eukaryote Saccharomyces cerevisiae.

The formation of pseudouridine and 5-methyluridine (m5U) in the T-arm of transfer RNAs (tRNAs) is near-universally conserved. These two modifications are formed in Escherichia coli by the pseudouridine synthase TruB and the S-adenosylmethionine-dependent methyltransferase TrmA, respectively. In this thesis, I investigate the function and mechanisms of these two tRNA modifying enzymes. First, in vitro and in vivo analysis of TruB reveals that this enzyme is acting as a tRNA chaperone which proves a long outstanding hypothesis. Secondly, characterization of ligand binding by TrmA shows that binding is cooperative and disruption of tRNA elbow region tertiary interactions by TrmA is essential for efficient tRNA binding and catalysis, leading to future analysis. In conclusion, my studies further our understanding of the mechanism and function of tRNA modifications and modifying enzymes, as well as shed light on why all cells invest substantial resources into fine-tuning the chemical composition of tRNAs.

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 forma

Cbf5 is a pseudouridine synthase that usually acts in a guide RNA-dependent manner as part of H/ACA small ribonucleoproteins; however archaeal Cbf5 can also act independently of guide RNA in modifying uridine 55 in tRNA. This guide-independent activity of Cbf5 is enhanced by proteins Nop10 and Gar1 which are also found in H/ACA small ribonucleoproteins. Here, we analyzed the specific contribution of Nop10 and Gar1 for Cbf5-catalyzed pseudouridylation of tRNA. Interestingly, both Nop10 and Gar1 not only increase Cbf5’s affinity for tRNA, but they also directly enhance Cbf5’s catalytic activity by increasing the kcat of the reaction. In contrast to the guide RNA-dependent reaction, Gar1 is not involved in product release after tRNA modification. These results in conjunction with structural information suggest that Nop10 and Gar1 stabilize Cbf5 in its active conformation; we hypothesize that this might also be true for guide-RNA dependent pseudouridine formation by Cbf5.

Pseudouridine synthases catalyze formation of the most abundant modification of functional RNAs by site-specifically isomerizing uridines to pseudouridines. While the structure and substrate specificity of these enzymes have been studied in detail, the kinetic and the catalytic mechanism of pseudouridine synthases remain unknown. Here, the first pre-steady-state kinetic analysis of three Escherichia coli pseudouridine synthases is presented. A novel stopped-flow absorbance assay revealed that substrate tRNA binding by TruB takes place in two steps with an overall rate of 6 sec 1. In order to observe catalysis of pseudouridine formation directly, the traditional tritium release assay was adapted for the quench-flow technique, allowing, for the first time, observation of a single round of pseudouridine formation. Thereby, the single-round rate constant of pseudouridylation (kC) by TruB was determined to be 0.5 sec 1. This rate constant is similar to the kcat obtained under multiple-turnover conditions in steady-state experiments, indicating that catalysis is the rate-limiting step for TruB. In order to investigate if pseudouridine synthases are characterized by slow catalysis in general, the rapid kinetic quench-flow analysis was also performed with two other E. coli enzymes, RluA and TruA, which displayed rate constants of pseudouridine formation of 0.7 and 0.35 sec 1, respectively. Hence, uniformly slow catalysis might be a general feature of pseudouridine synthases that share a conserved catalytic domain and supposedly use the same catalytic mechanism.

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.