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Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling
Published on: October 7, 2021
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Comprehensive sequence-to-function mapping of cofactor-dependent RNA catalysis in the glmS ribozyme.
Johan O L Andreasson1,2, Andrew Savinov3,4, Steven M Block5,6
1Department of Genetics, Stanford University, Stanford, CA, 94305, USA.
Nature Communications
|April 5, 2020
Summary
We developed an RNA-array assay to measure biomolecular activity, revealing how mutations affect the glmS ribozyme
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Biology
Background:
- Understanding RNA sequence-function relationships is crucial for molecular biology.
- Quantitative measurements across RNA sequence space can advance this understanding.
- The glmS ribozyme riboswitch is a model system for studying ligand-dependent RNA activity.
Purpose of the Study:
- To develop and apply a novel RNA-array assay for high-throughput, quantitative measurement of RNA activity.
- To systematically investigate the impact of mutations on the glmS ribozyme's self-cleavage reaction.
- To map sequence-function relationships and understand the structural basis of ribozyme activity.
Main Methods:
- Development of a high-throughput RNA-array assay for quantitative biomolecular activity measurements.
- Measurement of cleavage rates for all single and double mutants of the glmS ribozyme.
- Determination of kinetic parameters (kcat and KM) for active ribozyme variants across varying ligand concentrations.
Main Results:
- Systematic measurement of kinetic parameters for a comprehensive set of glmS ribozyme mutants.
- Evidence suggesting evolutionary conservation is driven by the maintenance of cleavage rates.
- Identification of specific tertiary interactions and their catalytic consequences through mutational analysis.
Conclusions:
- The developed RNA-array assay enables large-scale quantitative analysis of RNA sequence-function.
- Mutational analysis provides a detailed structural and functional map of the glmS ribozyme.
- Understanding sequence-function relationships can reveal mechanisms of structural adaptation in RNA molecules.
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