Wieden-Kothe, Ute

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https://opus.uleth.ca/handle/10133/4743

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Browsing Wieden-Kothe, Ute by Subject "RNA modification"

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    Contribution of two conserved histidines to the dual activity of archael RNA guide-dependent and -independent pseudouridine synthase Cbf5
    (Cold Spring Harbor Laboratory Press, 2015) Tillault, Anne-Sophie; Fourmann, Jean-Baptiste; Loegler, Christine; Wieden, Hans-Joachim; Wieden-Kothe, Ute; Charpentier, Bruno
    In all organisms, several distinct stand-alone pseudouridine synthase (PUS) family enzymes are expressed to isomerizeuridine into pseudouridine (Ψ) by specific recognition of RNAs. In addition, Ψs are generated in Archaea and Eukaryotes by PUS enzymes which are organized as ribonucleoprotein particles (RNP)—the box H/ACA s/snoRNPs. For this modification system, a unique TruB-like catalytic PUS subunit is associated with various RNA guides which specifically target and secure substrate RNAs by base-pairing. The archaeal Cbf5 PUS displays the special feature of exhibiting both RNA guide-dependent and -independent activities. Structures of substrate-bound TruB and H/ACA sRNP revealed the importance of histidines in positioning the target uridine in the active site. To analyze the respective role of H60 and H77, we have generated variants carrying alanine substitutions at these positions. The impact of the mutations was analyzed for unguided modifications U55 in tRNA and U2603 in 23S rRNA, and for activity of the box H/ACA Pab91 sRNP enzyme. H77 (H43 in TruB), but not H60, appeared to be crucial for the RNA guide-independent activity. In contrast to earlier suggestions, H60 was found to be noncritical for the activity of the H/ACA sRNP, but contributes together with H77 to the full activity of H/ACA sRNPs. The data suggest that a similar catalytic process was conserved in the two divergent pseudouridylation systems.
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    Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis
    (Cold Springs Harbor Laboratory Press, 2011) Wright, Jaden R.; Keffer-Wilkes, Laura C.; Dobing, Selina R.; Wieden-Kothe, Ute
    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.