OPUS: Open Ulethbridge Scholarship
Open ULeth Scholarship (OPUS) is the University of Lethbridge's open access research repository. It contains a collection of materials related to research and teaching produced by the academic community.
Self-archiving your research in OPUS is one way to meet Open Access policies of granting agencies. It is important to retain your final, post-peer-reviewed drafts for submission to OPUS, as this is often the only version publishers will allow to be archived. Click here for information on the U of L Open Access Policy.
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Oxidative stress damages rRNA inside the ribosome and differentially affects the catalytic center
(Oxford Academic, 2018) Willi, Jessica A.; Küpfer, Pascal; Evéquoz, Damien; Fernandez, Guillermo; Katz, Assaf; Leumann, Christian; Polacek, Norbert
Intracellular levels of reactive oxygen species (ROS) increase as a consequence of oxidative stress and represent a major source of damage to biomolecules. Due to its high cellular abundance RNA is more frequently the target for oxidative damage than DNA. Nevertheless the functional consequences of damage on stable RNA are poorly understood. Using a genome-wide approach, based on 8-oxo-guanosine immunoprecipitation, we present evidence that the most abundant non-coding RNA in a cell, the ribosomal RNA (rRNA), is target for oxidative nucleobase damage by ROS. Subjecting ribosomes to oxidative stress, we demonstrate that oxidized 23S rRNA inhibits the ribosome during protein biosynthesis. Placing single oxidized nucleobases at specific position within the ribosome's catalytic center by atomic mutagenesis resulted in markedly different functional outcomes. While some active site nucleobases tolerated oxidative damage well, oxidation at others had detrimental effects on protein synthesis by inhibiting different sub-steps of the ribosomal elongation cycle. Our data provide molecular insight into the biological consequences of RNA oxidation in one of the most central cellular enzymes and reveal mechanistic insight on the role of individual active site nucleobases during translation.
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Dynamic 23S rRNA modification ho5C2501 benefits Escherichia coli under oxidative stress
(Oxford Academic, 2021) Fasnacht, Michel; Gallo, Stefano; Sharma, Puneet; Himmelstob, Maximilian; Limbach, Patrick A.; Willi, Jessica A.; Polacek, Norbert
Post-transcriptional modifications are added to ribosomal RNAs (rRNAs) to govern ribosome biogenesis and to fine-tune protein biosynthesis. In Escherichia coli and related bacteria, RlhA uniquely catalyzes formation of a 5-hydroxycytidine (ho5C) at position 2501 of 23S rRNA. However, the molecular and biological functions as well as the regulation of ho5C2501 modification remain unclear. We measured growth curves with the modification-deficient ΔrlhA strain and quantified the extent of the modification during different conditions by mass spectrometry and reverse transcription. The levels of ho5C2501 in E. coli ribosomes turned out to be highly dynamic and growth phase-dependent, with the most effective hydroxylation yields observed in the stationary phase. We demonstrated a direct effect of ho5C2501 on translation efficiencies in vitro and in vivo. High ho5C2501 levels reduced protein biosynthesis which however turned out to be beneficial for E. coli for adapting to oxidative stress. This functional advantage was small under optimal conditions or during heat or cold shock, but becomes pronounced in the presence of hydrogen peroxide. Taken together, these data provided first functional insights into the role of this unique 23S rRNA modification for ribosome functions and bacterial growth under oxidative stress.
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Computationally-guided design and selection of high performing ribosomal active site mutants
(Oxford Academic, 2022) Kofman, Camila; Watkins, Andrew M.; Kim, Do Soon; Willi, Jessica A.; Wooldredge, Alexandra C.; Karim, Ashty S.; Das, Rhiju; Jewett, Michael C.
Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selection may serve as a powerful tool for rTNA engineering.
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Alternate conformational trajectories in ribosome translocation
(Public Library of Science, 2024) Alejo, Jose L.; Girodat, Dylan; Hammerling, Michael J.; Willi, Jessica A.; Jewett, MIchael C.; Engelhart, Aaron E.; Adamala, Katarzyna P.
Translocation in protein synthesis entails the efficient and accurate movement of the mRNA-[tRNA]2 substrate through the ribosome after peptide bond formation. An essential conformational change during this process is the swiveling of the small subunit head domain about two rRNA ‘hinge’ elements. Using iterative selection and molecular dynamics simulations, we derive alternate hinge elements capable of translocation in vitro and in vivo and describe their effects on the conformational trajectory of the EF-G-bound, translocating ribosome. In these alternate conformational pathways, we observe a diversity of swivel kinetics, hinge motions, three-dimensional head domain trajectories and tRNA dynamics. By finding alternate conformational pathways of translocation, we identify motions and intermediates that are essential or malleable in this process. These findings highlight the plasticity of protein synthesis and provide a more thorough understanding of the available sequence and conformational landscape of a central biological process.
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Peplau’s theory of interpersonal relations: application to asynchronous nursing education
(2022) Vogelsang, Laura
The COVID-19 pandemic has changed the course of nursing education delivery in many ways. Although programs have resumed in-person classes for much of the curriculum some online delivery continues. Peplau’s Theory of Interpersonal Relations is summarized and suggested as a way to maintain interpersonal relationship with students in the online setting. Proposition of the new role of ‘innovator’ for the nurse and an expanded definition of communication which fits with the online context are suggested. Application to asynchronous nursing education with consideration of modern-day teaching methodologies are discussed.
Throughout the 21st century the use of technology as a tool for communication has exploded. Whether through telephone, social media, or instant message, individuals increasingly use digital means to stay connected. At the height of the pandemic entire nursing programs were delivered online and connecting synchronously and asynchronously though a virtual means became the norm. Relationships were formed between students and instructors without an in-person meeting ever occurring. As many nursing programs return to in-person delivery some of the advantages of virtual clinical were realized and many program continue to offer virtual components (Seah et al., 2020). Several existing nursing theories offer guidance for forming effective nurse-client relationships. However, these theories fail to address the ‘technology’ component of communication and the impact of the lack of physical presence.
Hildegard Peplau (1909-1999) was a pioneer of the nurse-client relationship. Her practice-based theory emphasized looking at using every interaction as an opportunity for empowerment. The aim of this paper is to critically examine the philosophical underpinnings of Peplau’s Theory of Interpersonal Relations and evaluate the potential contributions it makes for supporting nursing educators to deliver clinical in a virtual setting. The definition of communication will be redefined for use in the virtual clinical setting. Further the writer will suggest an additional role of the nurse, the innovator role. This theory can potentially contribute to effective nursing education when applied in a virtual educational setting.