47 results on '"Kothe U"'
Search Results
2. Codon reading by tRNAAla with modified uridine in the wobble position
- Author
-
Kothe, U. and Rodnina, M.
- Published
- 2007
3. Anwendung der Six Sigma Strategie in der Fertigung von Schiffssektionen
- Author
-
Wanner, M.-C., Kothe, U., and Publica
- Subjects
Fertigungsprozeß ,Schiffbau ,Six Sigma ,Schiffssektion - Abstract
Prof. Dr.-Ing. M.C. Wanner und Dr.-Ing. U. Kothe stellen eine Strategie vor, mit der eine Stabilisierung des Fertigungsprozesses durch technologische Maßnahmen erreicht werden kann.
- Published
- 2005
4. An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
- Author
-
Friedt, J., primary, Leavens, F. M. V., additional, Mercier, E., additional, Wieden, H.-J., additional, and Kothe, U., additional
- Published
- 2013
- Full Text
- View/download PDF
5. Deltr: Digital embryo lineage tree reconstructor
- Author
-
Lou, X., primary, Kaster, F. O., additional, Lindner, M. S., additional, Kausler, B. X., additional, Kothe, U., additional, Hockendorf, B., additional, Wittbrodt, J., additional, Janicke, H., additional, and Hamprecht, F. A., additional
- Published
- 2011
- Full Text
- View/download PDF
6. Ilastik: Interactive learning and segmentation toolkit.
- Author
-
Sommer, C., Straehle, C., Kothe, U., and Hamprecht, F.A.
- Published
- 2011
- Full Text
- View/download PDF
7. On errors-in-variables regression with arbitrary covariance and its application to optical flow estimation.
- Author
-
Andres, B., Kondermann, C., Kondermann, D., Kothe, U., Hamprecht, F.A., and Garbe, C.S.
- Published
- 2008
- Full Text
- View/download PDF
8. Seroprevalence of Borrelia infection in occupational tick-exposed people in Bavaria (Germany)
- Author
-
Reimer, B., primary, Erbas, B., additional, Lobbichler, K., additional, Truckenbrodt, R., additional, Gartner-Kothe, U., additional, Kapeller, N., additional, Hansen, M., additional, Fingerle, V., additional, Wilske, B., additional, and Sonnenburg, F.v., additional
- Published
- 2002
- Full Text
- View/download PDF
9. Integrated edge and junction detection with the boundary tensor.
- Author
-
Kothe, U.
- Published
- 2003
- Full Text
- View/download PDF
10. ERSO-acquisition, reconstruction and simulation of real objects.
- Author
-
Diener, H., Kothe, U., Ristow, B., Schreyer, M., and Stelbe, U.
- Published
- 1998
- Full Text
- View/download PDF
11. Canada's contributions to RNA research: past, present, and future perspectives.
- Author
-
Lécuyer E, Sauvageau M, Kothe U, Unrau PJ, Damha MJ, Perreault J, Abou Elela S, Bayfield MA, Claycomb JM, and Scott MS
- Subjects
- Canada, Humans, SARS-CoV-2 genetics, Biomedical Research, COVID-19 epidemiology, RNA genetics
- Abstract
The field of RNA research has provided profound insights into the basic mechanisms modulating the function and adaption of biological systems. RNA has also been at the center stage in the development of transformative biotechnological and medical applications, perhaps most notably was the advent of mRNA vaccines that were critical in helping humanity through the Covid-19 pandemic. Unbeknownst to many, Canada boasts a diverse community of RNA scientists, spanning multiple disciplines and locations, whose cutting-edge research has established a rich track record of contributions across various aspects of RNA science over many decades. Through this position paper, we seek to highlight key contributions made by Canadian investigators to the RNA field, via both thematic and historical viewpoints. We also discuss initiatives underway to organize and enhance the impact of the Canadian RNA research community, particularly focusing on the creation of the not-for-profit organization RNA Canada ARN. Considering the strategic importance of RNA research in biology and medicine, and its considerable potential to help address major challenges facing humanity, sustained support of this sector will be critical to help Canadian scientists play key roles in the ongoing RNA revolution and the many benefits this could bring about to Canada., Competing Interests: The authors declare no conflicts of interest.
- Published
- 2024
- Full Text
- View/download PDF
12. RNA modifying enzymes shape tRNA biogenesis and function.
- Author
-
Schultz SK and Kothe U
- Subjects
- Humans, Protein Biosynthesis, Animals, Anticodon metabolism, Anticodon genetics, RNA, Transfer metabolism, RNA, Transfer genetics, RNA Processing, Post-Transcriptional
- Abstract
Transfer RNAs (tRNAs) are the most highly modified cellular RNAs, both with respect to the proportion of nucleotides that are modified within the tRNA sequence and with respect to the extraordinary diversity in tRNA modification chemistry. However, the functions of many different tRNA modifications are only beginning to emerge. tRNAs have two general clusters of modifications. The first cluster is within the anticodon stem-loop including several modifications essential for protein translation. The second cluster of modifications is within the tRNA elbow, and roles for these modifications are less clear. In general, tRNA elbow modifications are typically not essential for cell growth, but nonetheless several tRNA elbow modifications have been highly conserved throughout all domains of life. In addition to forming modifications, many tRNA modifying enzymes have been demonstrated or hypothesized to also play an important role in folding tRNA acting as tRNA chaperones. In this review, we summarize the known functions of tRNA modifying enzymes throughout the lifecycle of a tRNA molecule, from transcription to degradation. Thereby, we describe how tRNA modification and folding by tRNA modifying enzymes enhance tRNA maturation, tRNA aminoacylation, and tRNA function during protein synthesis, ultimately impacting cellular phenotypes and disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
13. Modifications in the T arm of tRNA globally determine tRNA maturation, function, and cellular fitness.
- Author
-
Schultz SK, Katanski CD, Halucha M, Peña N, Fahlman RP, Pan T, and Kothe U
- Subjects
- Protein Biosynthesis, Escherichia coli Proteins metabolism, Escherichia coli Proteins genetics, tRNA Methyltransferases metabolism, tRNA Methyltransferases genetics, RNA Processing, Post-Transcriptional, RNA, Transfer metabolism, RNA, Transfer genetics, Escherichia coli genetics, Escherichia coli metabolism
- Abstract
Almost all elongator tRNAs (Transfer RNAs) harbor 5-methyluridine 54 and pseudouridine 55 in the T arm, generated by the enzymes TrmA and TruB, respectively, in Escherichia coli. TrmA and TruB both act as tRNA chaperones, and strains lacking trmA or truB are outcompeted by wild type. Here, we investigate how TrmA and TruB contribute to cellular fitness. Deletion of trmA and truB in E. coli causes a global decrease in aminoacylation and alters other tRNA modifications such as acp
3 U47. While overall protein synthesis is not affected in Δ trmA and Δ truB strains, the translation of a subset of codons is significantly impaired. As a consequence, we observe translationally reduced expression of many specific proteins, that are either encoded with a high frequency of these codons or that are large proteins. The resulting proteome changes are not related to a specific growth phenotype, but overall cellular fitness is impaired upon deleting trmA and truB in accordance with a general protein synthesis impact. In conclusion, we demonstrate that universal modifications of the tRNA T arm are critical for global tRNA function by enhancing tRNA maturation, tRNA aminoacylation, and translation, thereby improving cellular fitness irrespective of the growth conditions which explains the conservation of trmA and truB ., Competing Interests: Competing interests statement:The authors declare no competing interest.- Published
- 2024
- Full Text
- View/download PDF
14. Amortized Bayesian Model Comparison With Evidential Deep Learning.
- Author
-
Radev ST, D'Alessandro M, Mertens UK, Voss A, Kothe U, and Burkner PC
- Abstract
Comparing competing mathematical models of complex processes is a shared goal among many branches of science. The Bayesian probabilistic framework offers a principled way to perform model comparison and extract useful metrics for guiding decisions. However, many interesting models are intractable with standard Bayesian methods, as they lack a closed-form likelihood function or the likelihood is computationally too expensive to evaluate. In this work, we propose a novel method for performing Bayesian model comparison using specialized deep learning architectures. Our method is purely simulation-based and circumvents the step of explicitly fitting all alternative models under consideration to each observed dataset. Moreover, it requires no hand-crafted summary statistics of the data and is designed to amortize the cost of simulation over multiple models, datasets, and dataset sizes. This makes the method especially effective in scenarios where model fit needs to be assessed for a large number of datasets, so that case-based inference is practically infeasible. Finally, we propose a novel way to measure epistemic uncertainty in model comparison problems. We demonstrate the utility of our method on toy examples and simulated data from nontrivial models from cognitive science and single-cell neuroscience. We show that our method achieves excellent results in terms of accuracy, calibration, and efficiency across the examples considered in this work. We argue that our framework can enhance and enrich model-based analysis and inference in many fields dealing with computational models of natural processes. We further argue that the proposed measure of epistemic uncertainty provides a unique proxy to quantify absolute evidence even in a framework which assumes that the true data-generating model is within a finite set of candidate models.
- Published
- 2023
- Full Text
- View/download PDF
15. Molecular mechanism of tRNA binding by the Escherichia coli N7 guanosine methyltransferase TrmB.
- Author
-
Schultz SK, Meadows K, and Kothe U
- Subjects
- Guanosine, RNA, Transfer metabolism, Escherichia coli metabolism, tRNA Methyltransferases chemistry
- Abstract
Among the large and diverse collection of tRNA modifications, 7-methylguanosine (m
7 G) is frequently found in the tRNA variable loop at position 46. This modification is introduced by the TrmB enzyme, which is conserved in bacteria and eukaryotes. However, the molecular determinants and the mechanism for tRNA recognition by TrmB are not well understood. Complementing the report of various phenotypes for different organisms lacking TrmB homologs, we report here hydrogen peroxide sensitivity for the Escherichia coli ΔtrmB knockout strain. To gain insight into the molecular mechanism of tRNA binding by E. coli TrmB in real time, we developed a new assay based on introducing a 4-thiouridine modification at position 8 of in vitro transcribed tRNAPhe enabling us to fluorescently label this unmodified tRNA. Using rapid kinetic stopped-flow measurements with this fluorescent tRNA, we examined the interaction of WT and single substitution variants of TrmB with tRNA. Our results reveal the role of S-adenosylmethionine for rapid and stable tRNA binding, the rate-limiting nature of m7 G46 catalysis for tRNA release, and the importance of residues R26, T127, and R155 across the entire surface of TrmB for tRNA binding., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
- Full Text
- View/download PDF
16. Fluorescent labeling of tRNA for rapid kinetic interaction studies with tRNA-binding proteins.
- Author
-
Schultz SK and Kothe U
- Subjects
- Nucleotides metabolism, Fluorescent Dyes metabolism, Fluoresceins, Carrier Proteins, RNA, Transfer metabolism
- Abstract
Transfer RNA (tRNA) plays a critical role during translation and interacts with numerous proteins during its biogenesis, functional cycle and degradation. In particular, tRNA is extensively post-transcriptionally modified by various tRNA modifying enzymes which each target a specific nucleotide at different positions within tRNAs to introduce different chemical modifications. Fluorescent assays can be used to study the interaction between a protein and tRNA. Moreover, rapid mixing fluorescence stopped-flow assays provide insights into the kinetics of the tRNA-protein interaction in order to elucidate the tRNA binding mechanism for the given protein. A prerequisite for these studies is a fluorescently labeled molecule, such as fluorescent tRNA, wherein a change in fluorescence occurs upon protein binding. In this chapter, we discuss the utilization of tRNA modifications in order to introduce fluorophores at particular positions within tRNAs. Particularly, we focus on in vitro thiolation of a uridine at position 8 within tRNAs using the tRNA modification enzyme ThiI, followed by labeling of the thiol group with fluorescein. As such, this fluorescently labeled tRNA is primarily unmodified, with the exception of the thiolation modification to which the fluorophore is attached, and can be used as a substrate to study the binding of different tRNA-interacting factors. Herein, we discuss the example of studying the tRNA binding mechanism of the tRNA modifying enzymes TrmB and DusA using internally fluorescein-labeled tRNA., (Copyright © 2023. Published by Elsevier Inc.)
- Published
- 2023
- Full Text
- View/download PDF
17. Multi-wavelength analytical ultracentrifugation of biopolymer mixtures and interactions.
- Author
-
Henrickson A, Gorbet GE, Savelyev A, Kim M, Hargreaves J, Schultz SK, Kothe U, and Demeler B
- Subjects
- Biopolymers, Ultracentrifugation methods, Hydrodynamics
- Abstract
Multi-wavelength analytical ultracentrifugation (MW-AUC) is a recent development made possible by new analytical ultracentrifuge optical systems. MW-AUC extends the basic hydrodynamic information content of AUC and provides access to a wide range of new applications for biopolymer characterization, and is poised to become an essential analytical tool to study 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 experimental investigations where the benefit of the added spectral dimension provides critical information that is not accessible, and impossible to resolve with traditional AUC methods. We demonstrate the improvements in resolution and information content obtained by this technique compared to traditional single- or dual-wavelength approaches, and discuss experimental design considerations and limitations of the method. We further address the advantages and disadvantages of the two MW optical systems available today, and the differences in data analysis strategies between the two systems., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
18. BayesFlow: Learning Complex Stochastic Models With Invertible Neural Networks.
- Author
-
Radev ST, Mertens UK, Voss A, Ardizzone L, and Kothe U
- Subjects
- Bayes Theorem, Learning, Neural Networks, Computer
- Abstract
Estimating the parameters of mathematical models is a common problem in almost all branches of science. However, this problem can prove notably difficult when processes and model descriptions become increasingly complex and an explicit likelihood function is not available. With this work, we propose a novel method for globally amortized Bayesian inference based on invertible neural networks that we call BayesFlow. The method uses simulations to learn a global estimator for the probabilistic mapping from observed data to underlying model parameters. A neural network pretrained in this way can then, without additional training or optimization, infer full posteriors on arbitrarily many real data sets involving the same model family. In addition, our method incorporates a summary network trained to embed the observed data into maximally informative summary statistics. Learning summary statistics from data makes the method applicable to modeling scenarios where standard inference techniques with handcrafted summary statistics fail. We demonstrate the utility of BayesFlow on challenging intractable models from population dynamics, epidemiology, cognitive science, and ecology. We argue that BayesFlow provides a general framework for building amortized Bayesian parameter estimation machines for any forward model from which data can be simulated.
- Published
- 2022
- Full Text
- View/download PDF
19. Synergistic interaction network between the snR30 RNP, Utp23, and ribosomal RNA during ribosome synthesis.
- Author
-
Vos TJ and Kothe U
- Subjects
- RNA, Ribosomal, 18S chemistry, Ribosomes genetics, Ribosomes metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Nuclear Proteins genetics, Nuclear Proteins metabolism, RNA Precursors genetics, RNA Precursors metabolism, Ribonucleoproteins metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
snR30/U17 is a highly conserved H/ACA RNA that is required for maturation of the small ribosomal subunit in eukaryotes. By base-pairing to the expansion segment 6 (ES6) of 18S ribosomal RNA (rRNA), the snR30 H/ACA Ribonucleoprotein (RNP) indirectly facilitates processing of the precursor rRNA (pre-rRNA) together with other proteins such as Utp23 and other RNAs acting as ribosome assembly factors. However, the details of the molecular interaction network of snR30 and its binding partners and how these interactions contribute to pre-rRNA processing remains unknown. Here, we report the in vitro reconstitution of a Saccharomyces cerevisiae snR30 RNP and quantitative characterization of the interactions of snR30, H/ACA proteins, the Utp23 protein and ES6 of the 18S rRNA. The snR30 RNA is bound tightly by both H/ACA proteins and Utp23. We dissected the importance of different 18S rRNA regions for snR30 RNP binding and demonstrated that the snR30 complex is tightly anchored on the pre-rRNA through base-pairing to ES6 whereas other reported rRNA binding sites do not contribute to the affinity of the snR30 RNP. On its own, the ribosome assembly factor Utp23 binds in a tight, but unspecific manner to RNA. However, in complex with the snR30 RNP, Utp23 increases the affinity of the RNP for rRNA revealing synergies between snR30 RNP and Utp23 which are enhancing specificity and affinity for rRNA, respectively. Together, these findings provide mechanistic insights how the snR30 RNP and Utp23 cooperate to interact tightly and specifically with rRNA during the early stages of ribosome biogenesis.
- Published
- 2022
- Full Text
- View/download PDF
20. The human pseudouridine synthase PUS7 recognizes RNA with an extended multi-domain binding surface.
- Author
-
Guegueniat J, Halabelian L, Zeng H, Dong A, Li Y, Wu H, Arrowsmith CH, and Kothe U
- Subjects
- Binding Sites, Humans, Intramolecular Transferases metabolism, Molecular Docking Simulation, Protein Binding, RNA, Transfer chemistry, Intramolecular Transferases chemistry, RNA, Transfer metabolism
- Abstract
The human pseudouridine synthase PUS7 is a versatile RNA modification enzyme targeting many RNAs thereby playing a critical role in development and brain function. Whereas all target RNAs of PUS7 share a consensus sequence, additional recognition elements are likely required, and the structural basis for RNA binding by PUS7 is unknown. Here, we characterize the structure-function relationship of human PUS7 reporting its X-ray crystal structure at 2.26 Å resolution. Compared to its bacterial homolog, human PUS7 possesses two additional subdomains, and structural modeling studies suggest that these subdomains contribute to tRNA recognition through increased interactions along the tRNA substrate. Consistent with our modeling, we find that all structural elements of tRNA are required for productive interaction with PUS7 as the consensus sequence of target RNA alone is not sufficient for pseudouridylation by human PUS7. Moreover, PUS7 binds several, non-modifiable RNAs with medium affinity which likely enables PUS7 to screen for productive RNA substrates. Following tRNA modification, the product tRNA has a significantly lower affinity for PUS7 facilitating its dissociation. Taken together our studies suggest a combination of structure-specific and sequence-specific RNA recognition by PUS7 and provide mechanistic insight into its function., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2021
- Full Text
- View/download PDF
21. The Mutex Watershed and its Objective: Efficient, Parameter-Free Graph Partitioning.
- Author
-
Wolf S, Bailoni A, Pape C, Rahaman N, Kreshuk A, Kothe U, and Hamprecht FA
- Abstract
Image partitioning, or segmentation without semantics, is the task of decomposing an image into distinct segments, or equivalently to detect closed contours. Most prior work either requires seeds, one per segment; or a threshold; or formulates the task as multicut / correlation clustering, an NP-hard problem. Here, we propose an efficient algorithm for graph partitioning, the "Mutex Watershed". Unlike seeded watershed, the algorithm can accommodate not only attractive but also repulsive cues, allowing it to find a previously unspecified number of segments without the need for explicit seeds or a tunable threshold. We also prove that this simple algorithm solves to global optimality an objective function that is intimately related to the multicut / correlation clustering integer linear programming formulation. The algorithm is deterministic, very simple to implement, and has empirically linearithmic complexity. When presented with short-range attractive and long-range repulsive cues from a deep neural network, the Mutex Watershed gives the best results currently known for the competitive ISBI 2012 EM segmentation benchmark.
- Published
- 2021
- Full Text
- View/download PDF
22. H/ACA Small Ribonucleoproteins: Structural and Functional Comparison Between Archaea and Eukaryotes.
- Author
-
Czekay DP and Kothe U
- Abstract
During ribosome synthesis, ribosomal RNA is modified through the formation of many pseudouridines and methylations which contribute to ribosome function across all domains of life. In archaea and eukaryotes, pseudouridylation of rRNA is catalyzed by H/ACA small ribonucleoproteins (sRNPs) utilizing different H/ACA guide RNAs to identify target uridines for modification. H/ACA sRNPs are conserved in archaea and eukaryotes, as they share a common general architecture and function, but there are also several notable differences between archaeal and eukaryotic H/ACA sRNPs. Due to the higher protein stability in archaea, we have more information on the structure of archaeal H/ACA sRNPs compared to eukaryotic counterparts. However, based on the long history of yeast genetic and other cellular studies, the biological role of H/ACA sRNPs during ribosome biogenesis is better understood in eukaryotes than archaea. Therefore, this review provides an overview of the current knowledge on H/ACA sRNPs from archaea, in particular their structure and function, and relates it to our understanding of the roles of eukaryotic H/ACA sRNP during eukaryotic ribosome synthesis and beyond. Based on this comparison of our current insights into archaeal and eukaryotic H/ACA sRNPs, we discuss what role archaeal H/ACA sRNPs may play in the formation of ribosomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Czekay and Kothe.)
- Published
- 2021
- Full Text
- View/download PDF
23. Revisiting tRNA chaperones: New players in an ancient game.
- Author
-
Porat J, Kothe U, and Bayfield MA
- Abstract
tRNAs undergo an extensive maturation process including post-transcriptional modifications that influence secondary and tertiary interactions. Precursor and mature tRNAs lacking key modifications are often recognized as aberrant and subsequently targeted for decay, illustrating the importance of modifications in promoting structural integrity. tRNAs also rely on tRNA chaperones to promote the folding of misfolded substrates into functional conformations. The best characterized tRNA chaperone is the La protein, which interacts with nascent RNA polymerase III transcripts to promote folding and offers protection from exonucleases. More recently, certain tRNA modification enzymes have also been demonstrated to possess tRNA folding activity distinct from their catalytic activity, suggesting that they may act as tRNA chaperones. In this review, we will discuss pioneering studies relating post-transcriptional modification to tRNA stability and decay pathways, present recent advances into the mechanism by which the RNA chaperone La assists pre-tRNA maturation, and summarize emerging research directions aimed at characterizing modification enzymes as tRNA chaperones. Together, these findings shed light on the importance of tRNA folding and how tRNA chaperones, in particular, increase the fraction of nascent pre-tRNAs that adopt a folded, functional conformation., (Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2021
- Full Text
- View/download PDF
24. Partially modified tRNAs for the study of tRNA maturation and function.
- Author
-
Schultz SK and Kothe U
- Subjects
- Ribosomes metabolism, RNA Processing, Post-Transcriptional, RNA, Transfer genetics, RNA, Transfer metabolism
- Abstract
Transfer RNA (tRNA) is the most highly and diversely modified class of RNA in all domains of life. However, we still have only a limited understanding of the concerted action of the many enzymes that modify tRNA during tRNA maturation and the synergistic functions of tRNA modifications for protein synthesis. Here, we describe the preparation of in vitro transcribed tRNAs with a partial set of defined modifications and the use of partially modified tRNAs in biochemical assays. By comparing the affinity and activity of tRNA modification enzymes for partially modified and unmodified tRNAs, we gain insight into the preferred pathways of tRNA maturation. Additionally, partially modified tRNAs will be highly useful to investigate the importance of tRNA modifications for tRNA function during translation including the interaction with aminoacyl-tRNA synthases, translation factors and the ribosome. Thereby, the methods described here lay the foundation for understanding the mechanistic function of tRNA modifications., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
25. Assaying the Molecular Determinants and Kinetics of RNA Pseudouridylation by H/ACA snoRNPs and Stand-Alone Pseudouridine Synthases.
- Author
-
Czekay DP, Schultz SK, and Kothe U
- Subjects
- Escherichia coli genetics, Kinetics, RNA Processing, Post-Transcriptional genetics, Saccharomyces cerevisiae genetics, RNA, Guide, CRISPR-Cas Systems, Intramolecular Transferases genetics, Pseudouridine genetics, RNA genetics, Ribonucleoproteins, Small Nucleolar genetics
- Abstract
Posttranscriptional modifications of RNA play an important role in promoting the maturation and functional diversity of many RNA species. Accordingly, understanding the enzymes and mechanisms that underlie RNA modifications is an important aspect in advancing our knowledge of the continually expanding RNA modification field. However, of the more than 160 currently identified RNA modifications, a large portion remains without quantitative detection assays for their biochemical characterization. Here, we describe the tritium release assay as a convenient tool allowing for the quantitative assessment of in vitro RNA pseudouridylation by stand-alone or box H/ACA RNA-guided pseudouridine synthases. This assay enables quantification of RNA pseudouridylation over a time course to effectively compare pseudouridylation activity between different substrates and/or different recombinant enzymes as well as to determine kinetic parameters. With the help of a quench-flow apparatus, the tritium release assay can be adapted for rapid kinetic measurements under single-turnover conditions to dissect reaction mechanisms. As examples, we show the formation of pseudouridines by a reconstituted Saccharomyces cerevisiae H/ACA small ribonucleoprotein (snoRNP) and an Escherichia coli stand-alone pseudouridine synthase.
- Published
- 2021
- Full Text
- View/download PDF
26. snR30/U17 Small Nucleolar Ribonucleoprotein: A Critical Player during Ribosome Biogenesis.
- Author
-
Vos TJ and Kothe U
- Subjects
- Cell Nucleolus genetics, Humans, RNA, Ribosomal, 18S genetics, Saccharomyces cerevisiae genetics, RNA Precursors genetics, RNA, Small Nucleolar genetics, Ribonucleoproteins, Small Nucleolar genetics, Ribosomes genetics
- Abstract
The small nucleolar RNA snR30 (U17 in humans) plays a unique role during ribosome synthesis. Unlike most members of the H/ACA class of guide RNAs, the small nucleolar ribonucleoprotein (snoRNP) complex assembled on snR30 does not direct pseudouridylation of ribosomal RNA (rRNA), but instead snR30 is critical for 18S rRNA processing during formation of the small subunit (SSU) of the ribosome. Specifically, snR30 is essential for three pre-rRNA cleavages at the A
0 /01, A1 /1, and A2 /2a sites in yeast and humans, respectively. Accordingly, snR30 is the only essential H/ACA guide RNA in yeast. Here, we summarize our current knowledge about the interactions and functions of snR30, discuss what remains to be elucidated, and present two non-exclusive hypotheses on the possible molecular function of snR30 during ribosome biogenesis. First, snR30 might be responsible for recruiting other proteins including endonucleases to the SSU processome. Second, snR30 may contribute to the refolding of pre-rRNA into a required conformation that serves as a checkpoint during ribosome biogenesis facilitating pre-rRNA cleavage. In both scenarios, the snR30 snoRNP may have scaffolding and RNA chaperoning activity. In conclusion, the snR30 snoRNP is a crucial player with an unknown molecular mechanism during ribosome synthesis, posing many interesting future research questions.- Published
- 2020
- Full Text
- View/download PDF
27. tRNA elbow modifications affect the tRNA pseudouridine synthase TruB and the methyltransferase TrmA.
- Author
-
Schultz SK and Kothe U
- Subjects
- Escherichia coli genetics, Intramolecular Transferases genetics, Pseudouridine genetics, RNA, Transfer genetics, tRNA Methyltransferases genetics
- Abstract
tRNAs constitute the most highly modified class of RNA. Every tRNA contains a unique set of modifications, and Ψ55, m
5 U54, and m7 G46 are frequently found within the elbow of the tRNA structure. Despite the abundance of tRNA modifications, we are only beginning to understand the orchestration of modification enzymes during tRNA maturation. Here, we investigated whether pre-existing modifications impact the binding affinity or catalysis by tRNA elbow modification enzymes. Specifically, we focused on the Escherichia coli enzymes TruB, TrmA, and TrmB which generate Ψ55, m5 U54, and m7 G46, respectively. tRNAs containing a single modification were prepared, and the binding and activity preferences of purified E. coli TrmA, TruB, and TrmB were examined in vitro. TruB preferentially binds and modifies unmodified tRNA. TrmA prefers to modify unmodified tRNA, but binds most tightly to tRNA that already contains Ψ55. In contrast, binding and modification by TrmB is insensitive to the tRNA modification status. Our results suggest that TrmA and TruB are likely to act on mostly unmodified tRNA precursors during the early stages of tRNA maturation whereas TrmB presumably acts on later tRNA intermediates that are already partially modified. In conclusion, we uncover the mechanistic basis for the preferred modification order in the E. coli tRNA elbow region., (© 2020 Schultz and Kothe; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)- Published
- 2020
- Full Text
- View/download PDF
28. The methyltransferase TrmA facilitates tRNA folding through interaction with its RNA-binding domain.
- Author
-
Keffer-Wilkes LC, Soon EF, and Kothe U
- Subjects
- Catalytic Domain, Protein Binding, RNA Folding, RNA, Transfer metabolism, RNA-Binding Motifs, tRNA Methyltransferases metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, RNA, Transfer chemistry, tRNA Methyltransferases chemistry
- Abstract
tRNAs are the most highly modified RNAs in all cells, and formation of 5-methyluridine (m5U) at position 54 in the T arm is a common RNA modification found in all tRNAs. The m5U modification is generated by the methyltransferase TrmA. Here, we test and prove the hypothesis that Escherichia coli TrmA has dual functions, acting both as a methyltransferase and as a tRNA chaperone. We identify two conserved residues, F106 and H125, in the RNA-binding domain of TrmA, which interact with the tRNA elbow and are critical for tRNA binding. Co-culture competition assays reveal that the catalytic activity of TrmA is important for cellular fitness, and that substitutions of F106 or H125 impair cellular fitness. We directly show that TrmA enhances tRNA folding in vitro independent of its catalytic activity. In conclusion, our study suggests that F106 and H125 in the RNA-binding domain of TrmA act as a wedge disrupting tertiary interactions between tRNA's D arm and T arm; this tRNA unfolding is the mechanistic basis for TrmA's tRNA chaperone activity. TrmA is the second tRNA modifying enzyme next to the pseudouridine synthase TruB shown to act as a tRNA chaperone supporting a functional link between RNA modification and folding., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2020
- Full Text
- View/download PDF
29. Parenting researchers-an invisible divide.
- Author
-
Roberts L, Kothe U, and Wieden HJ
- Subjects
- Female, Humans, Parenting, Research Personnel
- Abstract
The corona pandemic is an opportunity to rethink and revamp the academic career and reward system that consistently disadvantages parenting scientists and women., (© 2020 The Authors.)
- Published
- 2020
- Full Text
- View/download PDF
30. Proceedings of the 14th annual RiboWest conference: perspectives and outcome.
- Author
-
Thakor N, Kothe U, Wieden HJ, and Patel TR
- Subjects
- Biomedical Research, Canada, Humans, Research Personnel, RNA genetics, RNA metabolism
- Abstract
The RiboWest Conference brings together RNA researchers in Canada with the 2-fold goals of fostering internationally competitive RNA research and of training the next generation of scientists. The 14th Annual RiboWest conference (RiboWest 2018) was held at the University of Lethbridge (Lethbridge, Alberta) from June 10th to 13th, 2018. This meeting was focused on all major aspects of RNA research, ranging from understanding the cellular role of RNA, studying RNA interactions and structures, and employing them as a therapeutic tool. The invited keynote speakers (5) provided insights into the wide-range of RNA-based research. One of the unique features of this conference was that the majority of the oral presentations were given by the trainees (undergraduate/graduate students and postdoctoral researchers). Hosted by the Alberta RNA Research and Training Institute (ARRTI) at the University of Lethbridge as the leading center of RNA research in Western Canada, the RiboWest 2018 was well attended by researchers from across the country (>110 attendees in total). This conference proceedings editorial presents the overview of the conference, and briefly introduces articles published in this special issue of Biochemistry and Cell Biology .
- Published
- 2020
- Full Text
- View/download PDF
31. Base-pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar ribonucleoproteins.
- Author
-
Kelly EK, Czekay DP, and Kothe U
- Subjects
- Kinetics, Substrate Specificity, RNA, Guide, CRISPR-Cas Systems, Base Pairing, Pseudouridine metabolism, Ribonucleoproteins, Small Nucleolar metabolism
- Abstract
H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the Saccharomyces cerevisiae H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base-pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide-substrate RNA pairs., (© 2019 Kelly et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2019
- Full Text
- View/download PDF
32. Molecular Determinants for 23S rRNA Recognition and Modification by the E. coli Pseudouridine Synthase RluE.
- Author
-
Tillault AS, Schultz SK, Wieden HJ, and Kothe U
- Subjects
- Binding Sites, Models, Molecular, Nucleic Acid Conformation, Protein Binding, Protein Conformation, Pseudouridine metabolism, RNA, Bacterial chemistry, RNA, Bacterial genetics, Substrate Specificity, Escherichia coli enzymology, Hydro-Lyases chemistry, Hydro-Lyases metabolism, RNA, Ribosomal, 23S chemistry, RNA, Ribosomal, 23S genetics
- Abstract
The isomerization of uridine to pseudouridine is the most common type of RNA modification found in RNAs across all domains of life and is performed by RNA-dependent and RNA-independent enzymes. The Escherichia coli pseudouridine synthase RluE acts as a stand-alone, highly specific enzyme forming the universally conserved pseudouridine at position 2457, located in helix 89 (H89) of the 23S rRNA in the peptidyltransferase center. Here, we conduct a detailed structure-function analysis to determine the structural elements both in RluE and in 23S rRNA required for RNA-protein interaction and pseudouridine formation. We determined that RluE recognizes a large part of 23S rRNA comprising both H89 and the single-stranded flanking regions which explains the high substrate specificity of RluE. Within RluE, the target RNA is recognized through sequence-specific contacts with loop L7-8 as well as interactions with loop L1-2 and the flexible N-terminal region. We demonstrate that RluE is a faster pseudouridine synthase than other enzymes which likely enables it to act in the early stages of ribosome formation. In summary, our biochemical characterization of RluE provides detailed insight into the molecular mechanism of RluE forming a highly conserved pseudouridine during ribosome biogenesis., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
33. Efficient RNA pseudouridylation by eukaryotic H/ACA ribonucleoproteins requires high affinity binding and correct positioning of guide RNA.
- Author
-
Caton EA, Kelly EK, Kamalampeta R, and Kothe U
- Subjects
- Algorithms, Base Sequence, Binding, Competitive, Kinetics, Nuclear Proteins genetics, Nuclear Proteins metabolism, Protein Binding, RNA genetics, RNA, Small Nucleolar genetics, RNA, Small Nucleolar metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Ribonucleoproteins genetics, Ribonucleoproteins, Small Nucleolar genetics, Ribonucleoproteins, Small Nucleolar metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, RNA, Guide, CRISPR-Cas Systems, Pseudouridine metabolism, RNA metabolism, Ribonucleoproteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
H/ACA ribonucleoproteins (H/ACA RNPs) are responsible for introducing many pseudouridines into RNAs, but are also involved in other cellular functions. Utilizing a purified and reconstituted yeast H/ACA RNP system that is active in pseudouridine formation under physiological conditions, we describe here the quantitative characterization of H/ACA RNP formation and function. This analysis reveals a surprisingly tight interaction of H/ACA guide RNA with the Cbf5p-Nop10p-Gar1p trimeric protein complex whereas Nhp2p binds comparably weakly to H/ACA guide RNA. Substrate RNA is bound to H/ACA RNPs with nanomolar affinity which correlates with the GC content in the guide-substrate RNA base pairing. Both Nhp2p and the conserved Box ACA element in guide RNA are required for efficient pseudouridine formation, but not for guide RNA or substrate RNA binding. These results suggest that Nhp2p and the Box ACA motif indirectly facilitate loading of the substrate RNA in the catalytic site of Cbf5p by correctly positioning the upper and lower parts of the H/ACA guide RNA on the H/ACA proteins. In summary, this study provides detailed insight into the molecular mechanism of H/ACA RNPs., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2018
- Full Text
- View/download PDF
34. Eukaryotic stand-alone pseudouridine synthases - RNA modifying enzymes and emerging regulators of gene expression?
- Author
-
Rintala-Dempsey AC and Kothe U
- Subjects
- Animals, Disease Susceptibility, Humans, Intramolecular Transferases genetics, Multigene Family, RNA chemistry, RNA, Messenger chemistry, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Small Nuclear chemistry, RNA, Small Nuclear genetics, RNA, Small Nuclear metabolism, RNA, Transfer chemistry, RNA, Transfer genetics, RNA, Transfer metabolism, RNA, Untranslated chemistry, RNA, Untranslated genetics, RNA, Untranslated metabolism, Substrate Specificity, Eukaryotic Cells metabolism, Gene Expression Regulation, Intramolecular Transferases metabolism, RNA genetics, RNA metabolism
- Abstract
For a long time, eukaryotic stand-alone pseudouridine synthases (Pus enzymes) were neglected as non-essential enzymes adding seemingly simple modifications to tRNAs and small nuclear RNAs. Most studies were limited to the identification and initial characterization of the yeast Pus enzymes. However, recent transcriptome-wide mapping of pseudouridines in yeast and humans revealed pervasive modification of mRNAs and other non-coding RNAs by Pus enzymes which is dynamically regulated in response to cellular stress. Moreover, mutations in at least 2 genes encoding human Pus enzymes cause inherited diseases affecting muscle and brain function. Together, the recent findings suggest a broader-than-anticipated role of the Pus enzymes which are emerging as potential regulators of gene expression. In this review, we summarize the current knowledge on Pus enzymes, generate hypotheses regarding their cellular function and outline future areas of research of pseudouridine synthases.
- Published
- 2017
- Full Text
- View/download PDF
35. RNA modification enzyme TruB is a tRNA chaperone.
- Author
-
Keffer-Wilkes LC, Veerareddygari GR, and Kothe U
- Subjects
- Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Gene Knockout Techniques, Genes, Bacterial, Intramolecular Transferases chemistry, Intramolecular Transferases genetics, Models, Molecular, Molecular Chaperones chemistry, Molecular Chaperones genetics, Molecular Chaperones metabolism, Mutagenesis, Site-Directed, Nucleic Acid Conformation, Protein Domains, RNA Folding, RNA, Bacterial chemistry, RNA, Transfer chemistry, Escherichia coli Proteins metabolism, Intramolecular Transferases metabolism, RNA, Bacterial metabolism, RNA, Transfer metabolism
- Abstract
Cellular RNAs are chemically modified by many RNA modification enzymes; however, often the functions of modifications remain unclear, such as for pseudouridine formation in the tRNA TΨC arm by the bacterial tRNA pseudouridine synthase TruB. Here we test the hypothesis that RNA modification enzymes also act as RNA chaperones. Using TruB as a model, we demonstrate that TruB folds tRNA independent of its catalytic activity, thus increasing the fraction of tRNA that can be aminoacylated. By rapid kinetic stopped-flow analysis, we identified the molecular mechanism of TruB's RNA chaperone activity: TruB binds and unfolds both misfolded and folded tRNAs thereby providing misfolded tRNAs a second chance at folding. Previously, it has been shown that a catalytically inactive TruB variant has no phenotype when expressed in an Escherichia coli truB KO strain [Gutgsell N, et al. (2000) RNA 6(12):1870-1881]. However, here we uncover that E. coli strains expressing a TruB variant impaired in tRNA binding and in in vitro tRNA folding cannot compete with WT E. coli. Consequently, the tRNA chaperone activity of TruB is critical for bacterial fitness. In conclusion, we prove the tRNA chaperone activity of the pseudouridine synthase TruB, reveal its molecular mechanism, and demonstrate its importance for cellular fitness. We discuss the likelihood that other RNA modification enzymes are also RNA chaperones., Competing Interests: The authors declare no conflict of interest.
- Published
- 2016
- Full Text
- View/download PDF
36. Contribution of two conserved histidines to the dual activity of archaeal RNA guide-dependent and -independent pseudouridine synthase Cbf5.
- Author
-
Tillault AS, Fourmann JB, Loegler C, Wieden HJ, Kothe U, and Charpentier B
- Subjects
- Base Sequence, DNA Primers, Polymerase Chain Reaction, RNA, Archaeal chemistry, RNA, Archaeal metabolism, Substrate Specificity, Histidine physiology, Intramolecular Transferases metabolism, RNA, Archaeal physiology
- Abstract
In all organisms, several distinct stand-alone pseudouridine synthase (PUS) family enzymes are expressed to isomerize uridine 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 U(55) 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., (© 2015 Tillault et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2015
- Full Text
- View/download PDF
37. An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation.
- Author
-
Friedt J, Leavens FM, Mercier E, Wieden HJ, and Kothe U
- Subjects
- Amino Acid Substitution, Biocatalysis, Catalytic Domain, Intramolecular Transferases genetics, Intramolecular Transferases metabolism, Molecular Dynamics Simulation, RNA, Transfer metabolism, Static Electricity, Arginine chemistry, Aspartic Acid chemistry, Intramolecular Transferases chemistry, Pseudouridine metabolism
- Abstract
Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB's catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.
- Published
- 2014
- Full Text
- View/download PDF
38. tRNA binding, positioning, and modification by the pseudouridine synthase Pus10.
- Author
-
Kamalampeta R, Keffer-Wilkes LC, and Kothe U
- Subjects
- Catalysis, Circular Dichroism, Hydro-Lyases chemistry, Hydro-Lyases genetics, Kinetics, Protein Binding, Protein Interaction Domains and Motifs, Pseudouridine biosynthesis, Pyrococcus furiosus genetics, Pyrococcus furiosus metabolism, RNA, Transfer chemistry, Hydro-Lyases metabolism, RNA, Transfer genetics, RNA, Transfer metabolism
- Abstract
Pus10 is the most recently identified pseudouridine synthase found in archaea and higher eukaryotes. It modifies uridine 55 in the TΨC arm of tRNAs. Here, we report the first quantitative biochemical analysis of tRNA binding and pseudouridine formation by Pyrococcus furiosus Pus10. The affinity of Pus10 for both substrate and product tRNA is high (Kd of 30nM), and product formation occurs with a Km of 400nM and a kcat of 0.9s(-1). Site-directed mutagenesis was used to demonstrate that the thumb loop in the catalytic domain is important for efficient catalysis; we propose that the thumb loop positions the tRNA within the active site. Furthermore, a new catalytic arginine residue was identified (arginine 208), which is likely responsible for triggering flipping of the target uridine into the active site of Pus10. Lastly, our data support the proposal that the THUMP-containing domain, found in the N-terminus of Pus10, contributes to binding of tRNA. Together, our findings are consistent with the hypothesis that tRNA binding by Pus10 occurs through an induced-fit mechanism, which is a prerequisite for efficient pseudouridine formation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
39. Archaeal proteins Nop10 and Gar1 increase the catalytic activity of Cbf5 in pseudouridylating tRNA.
- Author
-
Kamalampeta R and Kothe U
- Subjects
- Enzyme Stability, Kinetics, Protein Binding, Pseudouridine biosynthesis, RNA, Small Untranslated, Archaeal Proteins chemistry, Intramolecular Transferases chemistry, Pyrococcus furiosus enzymology, RNA, Transfer chemistry, Ribonucleoproteins, Small Nucleolar chemistry
- Abstract
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 k(cat) 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.
- Published
- 2012
- Full Text
- View/download PDF
40. Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis.
- Author
-
Wright JR, Keffer-Wilkes LC, Dobing SR, and Kothe U
- Subjects
- Catalysis, Escherichia coli genetics, Gene Expression Regulation, Bacterial, Intramolecular Transferases genetics, Intramolecular Transferases isolation & purification, Kinetics, Protein Binding, Pseudouridine chemistry, Pseudouridine metabolism, RNA, Transfer metabolism, Escherichia coli enzymology, Intramolecular Transferases metabolism
- Abstract
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 (k(Ψ)) by TruB was determined to be 0.5 sec(-1). This rate constant is similar to the k(cat) 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.
- Published
- 2011
- Full Text
- View/download PDF
41. Codon reading by tRNAAla with modified uridine in the wobble position.
- Author
-
Kothe U and Rodnina MV
- Subjects
- Base Sequence, Guanosine Triphosphate metabolism, Hydrolysis, Kinetics, Molecular Sequence Data, Nucleic Acid Conformation, Peptides metabolism, RNA, Transfer, Ala chemistry, RNA, Transfer, Ala genetics, Uridine chemistry, Codon genetics, RNA, Transfer, Ala metabolism, Reading Frames genetics, Uridine metabolism
- Abstract
tRNAs reading four-codon families often have a modified uridine, cmo(5)U(34), at the wobble position of the anticodon. Here, we examine the effects on the decoding mechanism of a cmo(5)U modification in tRNA(1B)(Ala), anticodon C(36)G(35)cmo(5)U(34). tRNA(1B)(Ala) reads its cognate codons in a manner that is very similar to that of tRNA(Phe). As Ala codons are GC rich and Phe codons AU rich, this similarity suggests a uniform decoding mechanism that is independent of the GC content of the codon-anticodon duplex or the identity of the tRNA. The presence of cmo(5)U at the wobble position of tRNA(1B)(Ala) permits fairly efficient reading of non-Watson-Crick and nonwobble bases in the third codon position, e.g., the GCC codon. The ribosome accepts the C-cmo(5)U pair as an almost-correct base pair, unlike third-position mismatches, which lead to the incorporation of incorrect amino acids and are efficiently rejected.
- Published
- 2007
- Full Text
- View/download PDF
42. Delayed release of inorganic phosphate from elongation factor Tu following GTP hydrolysis on the ribosome.
- Author
-
Kothe U and Rodnina MV
- Subjects
- Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Guanosine Diphosphate metabolism, Kinetics, Models, Molecular, Protein Conformation, RNA, Messenger metabolism, RNA, Transfer, Amino Acyl metabolism, Guanosine Triphosphate metabolism, Peptide Elongation Factor Tu chemistry, Peptide Elongation Factor Tu metabolism, Phosphates metabolism, Ribosomes metabolism
- Abstract
The dissociation of inorganic phosphate (P(i)) following GTP hydrolysis is a key step determining the functional state of many GTPases. Here, the timing of P(i) release from elongation factor Tu (EF-Tu) and its implications for the function of EF-Tu on the ribosome were studied by rapid kinetic techniques. It was found that P(i) release from EF-Tu is >20-fold slower than GTP cleavage and limits the rate of the conformational switch of EF-Tu from the GTP- to the GDP-bound form. The point mutation Gly94Ala in the switch 2 region of EF-Tu abolished the delay in P(i) release, suggesting that P(i) release is controlled by the mobility of the switch 2 region with Gly94 acting as a pivot. The rate of P(i) release or the conformational switch of EF-Tu does not affect the selection of aminoacyl-tRNA on the ribosome. Rather, the slow P(i) release may be a consequence of the tight interaction of the switch regions of EF-Tu with the gamma-phosphate and the ribosome in the GTPase activated state of the factor.
- Published
- 2006
- Full Text
- View/download PDF
43. Single-step purification of specific tRNAs by hydrophobic tagging.
- Author
-
Kothe U, Paleskava A, Konevega AL, and Rodnina MV
- Subjects
- Amino Acids, Chromatography, High Pressure Liquid methods, Escherichia coli chemistry, Fluorenes, Hydrophobic and Hydrophilic Interactions, RNA, Bacterial isolation & purification, RNA, Transfer chemistry, RNA, Transfer, Ala isolation & purification, RNA, Transfer, Amino Acid-Specific isolation & purification, RNA, Transfer isolation & purification
- Published
- 2006
- Full Text
- View/download PDF
44. Control of phosphate release from elongation factor G by ribosomal protein L7/12.
- Author
-
Savelsbergh A, Mohr D, Kothe U, Wintermeyer W, and Rodnina MV
- Subjects
- Crystallography, X-Ray, Escherichia coli metabolism, Escherichia coli Proteins, Guanosine Triphosphate chemistry, Hydrolysis, Kinetics, Models, Biological, Models, Molecular, Mutation, Peptide Elongation Factor G metabolism, Phosphates chemistry, Plasmids metabolism, Protein Binding, Protein Transport, RNA, Messenger metabolism, RNA, Transfer chemistry, RNA, Transfer metabolism, Ribosomes chemistry, Ribosomes metabolism, Time Factors, Peptide Elongation Factor G physiology, Phosphates metabolism, Ribosomal Proteins chemistry, Ribosomal Proteins physiology
- Abstract
Ribosomal protein L7/12 is crucial for the function of elongation factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. Single conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G.GTP for the ribosome, suggesting an involvement of these residues in EF-G binding. The mutations did not appreciably affect rapid single-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorganic phosphate (Pi) from ribosome-bound EF-G.GDP.Pi was strongly inhibited and became rate-limiting for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaining the conformational coupling between EF-G and the ribosome for translocation and for timing the dissociation of the factor from the ribosome.
- Published
- 2005
- Full Text
- View/download PDF
45. Structural basis for the function of the ribosomal L7/12 stalk in factor binding and GTPase activation.
- Author
-
Diaconu M, Kothe U, Schlünzen F, Fischer N, Harms JM, Tonevitsky AG, Stark H, Rodnina MV, and Wahl MC
- Subjects
- Amino Acid Sequence, Binding Sites physiology, Cryoelectron Microscopy, Crystallography, X-Ray, Enzyme Activation physiology, Escherichia coli genetics, Escherichia coli ultrastructure, Models, Molecular, Molecular Sequence Data, Prokaryotic Initiation Factors metabolism, Protein Structure, Secondary physiology, Protein Structure, Tertiary physiology, Protein Subunits chemistry, Protein Subunits metabolism, RNA, Ribosomal metabolism, Ribosomal Protein L10, Ribosomal Proteins ultrastructure, Ribosomes genetics, Ribosomes ultrastructure, Thermotoga maritima genetics, Thermotoga maritima ultrastructure, Escherichia coli metabolism, GTP Phosphohydrolases metabolism, Ribosomal Proteins chemistry, Ribosomal Proteins metabolism, Ribosomes metabolism, Thermotoga maritima metabolism
- Abstract
The L7/12 stalk of the large subunit of bacterial ribosomes encompasses protein L10 and multiple copies of L7/12. We present crystal structures of Thermotoga maritima L10 in complex with three L7/12 N-terminal-domain dimers, refine the structure of an archaeal L10E N-terminal domain on the 50S subunit, and identify these elements in cryo-electron-microscopic reconstructions of Escherichia coli ribosomes. The mobile C-terminal helix alpha8 of L10 carries three L7/12 dimers in T. maritima and two in E. coli, in concordance with the different length of helix alpha8 of L10 in these organisms. The stalk is organized into three elements (stalk base, L10 helix alpha8-L7/12 N-terminal-domain complex, and L7/12 C-terminal domains) linked by flexible connections. Highly mobile L7/12 C-terminal domains promote recruitment of translation factors to the ribosome and stimulate GTP hydrolysis by the ribosome bound factors through stabilization of their active GTPase conformation.
- Published
- 2005
- Full Text
- View/download PDF
46. Recognition and selection of tRNA in translation.
- Author
-
Rodnina MV, Gromadski KB, Kothe U, and Wieden HJ
- Subjects
- Crystallography, RNA, Messenger genetics, RNA, Messenger metabolism, Ribosomes chemistry, Ribosomes physiology, Protein Biosynthesis physiology, RNA, Transfer, Amino Acyl metabolism
- Abstract
Aminoacyl-tRNA (aa-tRNA) is delivered to the ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. The stepwise movement of aa-tRNA from EF-Tu into the ribosomal A site entails a number of intermediates. The ribosome recognizes aa-tRNA through shape discrimination of the codon-anticodon duplex and regulates the rates of GTP hydrolysis by EF-Tu and aa-tRNA accommodation in the A site by an induced fit mechanism. Recent results of kinetic measurements, ribosome crystallography, single molecule FRET measurements, and cryo-electron microscopy suggest the mechanism of tRNA recognition and selection.
- Published
- 2005
- Full Text
- View/download PDF
47. Interaction of helix D of elongation factor Tu with helices 4 and 5 of protein L7/12 on the ribosome.
- Author
-
Kothe U, Wieden HJ, Mohr D, and Rodnina MV
- Subjects
- Amino Acid Substitution, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Binding Sites, Hydrophobic and Hydrophilic Interactions, Mutation, Peptide Elongation Factor Tu chemistry, Peptide Elongation Factor Tu genetics, Protein Binding, Protein Structure, Secondary, Ribosomal Proteins chemistry, Ribosomal Proteins genetics, Ribosomes metabolism, Static Electricity, Peptide Elongation Factor Tu metabolism, Protein Interaction Mapping, Ribosomal Proteins metabolism, Ribosomes chemistry
- Abstract
Elongation factor Tu (EF-Tu) promotes binding of aminoacyl-tRNA to the A site of the ribosome. Here, we report the effects of mutations in helix D of EF-Tu and in the C-terminal domain of L7/12 on the kinetics of A-site binding. Reaction rates were measured by stopped-flow and quench-flow techniques. The rates of A-site binding were decreased by mutations at positions 144, 145, 148, and 152 in helix D of EF-Tu as well as at positions 65, 66, 69, 70, 73, and 84 in helices 4 and 5 of L7/12. The effect was due primarily to the lower association rate constant of ternary complex binding to the ribosome. These results suggest that helix D of EF-Tu is involved in an initial transient contact with helices 4 and 5 of L7/12 that promotes ternary complex binding to the ribosome. By analogy to the interaction of helix D of EF-Tu with the N-terminal domain of EF-Ts, the contact area is likely to consist of a hydrophobic patch flanked by two salt-bridges.
- Published
- 2004
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.