Wieden-Kothe, Ute

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Browsing Wieden-Kothe, Ute by Subject "Cbf5"

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    Archael proteins Nop10 and Gar1 increase the catalytic activity of Cbf5 in pseudouridylating tRNA
    (Nature Publishing Group, 2012) Kamalampeta, Rajashekhar; Wieden-Kothe, Ute
    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.
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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.