Comprehensive sequence-to-function mapping of cofactor-dependent RNA catalysis in the glmS ribozyme

Johan O L Andreasson; Andrew Savinov; Steven M Block; William J Greenleaf

doi:10.1038/s41467-020-15540-1

Comprehensive sequence-to-function mapping of cofactor-dependent RNA catalysis in the glmS ribozyme

Nat Commun. 2020 Apr 3;11(1):1663. doi: 10.1038/s41467-020-15540-1.

Authors

Johan O L Andreasson^#^{1

2}, Andrew Savinov^#^{3

4}, Steven M Block^{5

6}, William J Greenleaf^{7

8

9}

Affiliations

¹ Department of Genetics, Stanford University, Stanford, CA, 94305, USA.
² Department of Biochemistry, Stanford University, Stanford, CA, 94305, USA.
³ Biophysics Program, Stanford University, Stanford, CA, 94305, USA.
⁴ Department of Genome Sciences, University of Washington, Seattle, WA, 98195, USA.
⁵ Department of Biology, Stanford University, Stanford, CA, 94305, USA. sblock@stanford.edu.
⁶ Department of Applied Physics, Stanford University, Stanford, CA, 94305, USA. sblock@stanford.edu.
⁷ Department of Genetics, Stanford University, Stanford, CA, 94305, USA. wjg@stanford.edu.
⁸ Department of Applied Physics, Stanford University, Stanford, CA, 94305, USA. wjg@stanford.edu.
⁹ Chan Zuckerberg Biohub, San Francisco, CA, USA. wjg@stanford.edu.

^# Contributed equally.

Abstract

Massively parallel, quantitative measurements of biomolecular activity across sequence space can greatly expand our understanding of RNA sequence-function relationships. We report the development of an RNA-array assay to perform such measurements and its application to a model RNA: the core glmS ribozyme riboswitch, which performs a ligand-dependent self-cleavage reaction. We measure the cleavage rates for all possible single and double mutants of this ribozyme across a series of ligand concentrations, determining k_cat and K_M values for active variants. These systematic measurements suggest that evolutionary conservation in the consensus sequence is driven by maintenance of the cleavage rate. Analysis of double-mutant rates and associated mutational interactions produces a structural and functional mapping of the ribozyme sequence, revealing the catalytic consequences of specific tertiary interactions, and allowing us to infer structural rearrangements that permit certain sequence variants to maintain activity.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Bacterial Proteins / chemistry
Bacterial Proteins / genetics*
Bacterial Proteins / metabolism
Consensus Sequence / genetics
Crystallography
Enzyme Assays
Evolution, Molecular*
High-Throughput Nucleotide Sequencing
Ligands
Mutation
Nucleic Acid Conformation
RNA, Catalytic / chemistry
RNA, Catalytic / genetics*
RNA, Catalytic / metabolism
Riboswitch / genetics*
Sequence Analysis, RNA
Structure-Activity Relationship

Substances

Bacterial Proteins
Ligands
RNA, Catalytic
Riboswitch
component S, glutamate mutase protein, Bacteria

Abstract

Publication types

MeSH terms

Substances

Grants and funding