RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes
- Boise State University, United States
- University of Edinburgh, United Kingdom
Abstract
Self-cleaving ribozymes are RNA molecules that catalyze the cleavage of their own phosphodiester backbones. These ribozymes are found in all domains of life and are also a tool for biotechnical and synthetic biology applications. Self-cleaving ribozymes are also an important model of sequence to function relationships for RNA because their small size simplifies synthesis of genetic variants and self-cleaving activity is an accessible readout of the functional consequence of the mutation. Here we used a high-throughput experimental approach to determine the relative activity for every possible single and double mutant of five self-cleaving ribozymes. From this data, we comprehensively identified non-additive effects between pairs of mutations (epistasis) for all five ribozymes. We analyzed how changes in activity and trends in epistasis map to the ribozyme structures. The variety of structures studied provided opportunities to observe several examples of common structural elements, and the data was collected under identical experimental conditions to enable direct comparison. Heat-map based visualization of the data revealed patterns indicating structural features of the ribozymes including paired regions, unpaired loops, non-canonical structures and tertiary structural contacts. The data also revealed signatures of functionally critical nucleotides involved in catalysis. The results demonstrate that the data sets provide structural information similar to chemical or enzymatic probing experiments, but with additional quantitative functional information. The large-scale data sets can be used for models predicting structure and function and for efforts to engineer self-cleaving ribozymes.
Data availability
Sequencing reads in FastQ format are available at ENA (PRJEB52899 and PRJEB51631). Sequences, activity data, and computer code is available at GitLab ( https://gitlab.com/bsu/biocompute-public/mut_12).
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RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymesEuropean Nucleotide Archive, PRJEB52899.
Article and author information
Author details
Funding
National Science Foundation (OIA-1738865)
- Eric J Hayden
National Science Foundation (OIA-1826801)
- Eric J Hayden
National Aeronautics and Space Administration (80NSSC17K0738)
- Eric J Hayden
Human Frontier Science Program (RGY0077/2019)
- Eric J Hayden
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Roberts et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes
eLife 12:e80360.
https://doi.org/10.7554/eLife.80360
Further reading
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
