Self-cleavage of the
            <i>glmS</i>
            ribozyme core is controlled by a fragile folding element

Savinov, Andrew; Block, Steven M.

doi:10.1073/pnas.1812122115

Cited by 16 publications

(8 citation statements)

References 36 publications

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“…4a, 5a). Consistent with our findings, P2.2 was previously proposed to play a key role in positioning the cleavage site and cofactor 17 , and more recently, the folding of this element was demonstrated directly to control catalysis 29 . We also found that the bottommost basepair of P2.2 (C2•G56) resulted in low activity (>850-fold below consensus) when changed to any alternative basepair (Fig.…”

Section: Double Mutant Cleavage Rates Reveal Mutational Interactionssupporting

confidence: 92%

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

et al. 2020

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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 liganddependent 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.

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Section: Double Mutant Cleavage Rates Reveal Mutational Interactionssupporting

confidence: 92%

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

et al. 2020

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“…Having established conditions and helper designs that yield strong protection and efficient site selectivity, we then proceeded to test the practical utility of the approach for acylation-based control of a tandem ribozyme. Although previous studies have described methods for control of a single catalytic RNA, − the use of a tandem ribozyme (TR) here offered the possibility to selectively control one RNA function relative to another within the same strand. An 81nt tandem hammerhead ribozyme with dual catalytic cores (3TR and 5TR) was produced by in vitro transcription and was reacted under site-selective conditions at their catalytic cores by the RAIL method under the standard conditions (Figures a, S6a; see SI for details).…”

Section: Resultsmentioning

confidence: 99%

Site-Selective RNA Functionalization via DNA-Induced Structure

2020

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Methods for RNA functionalization at specific sites are in high demand but remain a challenge, particularly for RNAs produced by transcription rather than by total synthesis. Recent studies have described acylimidazole reagents that react in high yields at 2′-OH groups stochastically at nonbase-paired regions, covering much of the RNA in scattered acyl esters. Localized reactions, if possible, could prove useful in many applications, providing functional handles at specific sites and sequences of the biopolymer. Here, we describe a DNA-directed strategy for in vitro functionalization of RNA at site-localized 2′-OH groups. The method, RNA Acylation at Induced Loops (RAIL), utilizes complementary helper DNA oligonucleotides that expose gaps or loops at selected positions while protecting the remainder in DNA-RNA duplexes. Reaction with an acylimidazole reagent is then carried out, providing high yields of 2′-OH conjugation at predetermined sites. Experiments reveal optimal helper oligodeoxynucleotide designs and conditions for the reaction, and tests of the approach are carried out to control localized ribozyme activities and to label RNAs with dual-color fluorescent dyes. The RAIL approach offers a simple and novel strategy for site-selective labeling and control of RNAs, potentially of any length and origin.

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“…This mode of transcription control was also reported for the Bacillus cereus crcB fluoride aptamer 18,47 . In contrast, dynamic pseudoknot folding and unfolding mediate translation control by the Faecalibacterium prausnitzii class III preQ 1 riboswitch 48 and RNA degradation control by the GlmS riboswitch 49 .…”

Section: Discussionmentioning

confidence: 99%

A ligand-gated strand displacement mechanism for ZTP riboswitch transcription control

et al. 2019

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Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure formation and function through transient intermediate folds. This process is particularly important for transcriptional riboswitches in which the formation of ligand-dependent structures during transcription regulates downstream gene expression. However, the intermediate structures that comprise cotranscriptional RNA folding pathways and the mechanisms that enable transit between them remain largely unknown. Here we determine the series of cotranscriptional folds and rearrangements that mediate antitermination by the Clostridium beijerinckii pfl ZTP riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate an internal RNA strand displacement process and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for pfl riboswitch antitermination and suggest general strategies by which nascent RNA molecules navigate cotranscriptional folding pathways.

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Self-cleavage of the glmS ribozyme core is controlled by a fragile folding element

Cited by 16 publications

References 36 publications

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

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

Site-Selective RNA Functionalization via DNA-Induced Structure

A ligand-gated strand displacement mechanism for ZTP riboswitch transcription control

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