Substrate binding and catalysis by the pseudouridine synthases RluA and TruB

xi, 122 leaves : ill. (some col.) ; 29 cmPseudouridine 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

Functional and mechanistic characterization of two tRNA modifying enzymes

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

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Sherpa Romeo green journal. Open access article. Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0) appliesPseudouridine 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.Ye

Identification of Fitness Determinants during Energy-Limited Growth Arrest in <i>Pseudomonas aeruginosa</i>

Microbial growth arrest can be triggered by diverse factors, one of which is energy limitation due to scarcity of electron donors or acceptors. Genes that govern fitness during energy-limited growth arrest and the extent to which they overlap between different types of energy limitation are poorly defined. In this study, we exploited the fact that Pseudomonas aeruginosa can remain viable over several weeks when limited for organic carbon (pyruvate) as an electron donor or oxygen as an electron acceptor. ATP values were reduced under both types of limitation, yet more severely in the absence of oxygen. Using transposon-insertion sequencing (Tn-seq), we identified fitness determinants in these two energy-limited states. Multiple genes encoding general functions like transcriptional regulation and energy generation were required for fitness during carbon or oxygen limitation, yet many specific genes, and thus specific activities, differed in their relevance between these states. For instance, the global regulator RpoS was required during both types of energy limitation, while other global regulators such as DksA and LasR were required only during carbon or oxygen limitation, respectively. Similarly, certain ribosomal and tRNA modifications were specifically required during oxygen limitation. We validated fitness defects during energy limitation using independently generated mutants of genes detected in our screen. Mutants in distinct functional categories exhibited different fitness dynamics: regulatory genes generally manifested a phenotype early, whereas genes involved in cell wall metabolism were required later. Together, these results provide a new window into how P. aeruginosa survives growth arrest

RNA-binding proteins in bacteria.

RNA-binding proteins (RBPs) are central to most if not all cellular processes, dictating the fate of virtually all RNA molecules in the cell. Starting with pioneering work on ribosomal proteins, studies of bacterial RBPs have paved the way for molecular studies of RNA-protein interactions. Work over the years has identified major RBPs that act on cellular transcripts at the various stages of bacterial gene expression and that enable their integration into post-transcriptional networks that also comprise small non-coding RNAs. Bacterial RBP research has now entered a new era in which RNA sequencing-based methods permit mapping of RBP activity in a truly global manner in vivo. Moreover, the soaring interest in understudied members of host-associated microbiota and environmental communities is likely to unveil new RBPs and to greatly expand our knowledge of RNA-protein interactions in bacteria