Rube Goldberg goes (ribo)nuclear? Molecular switches and sensors made from RNA

Silverman, Scott

doi:10.1261/rna.2200903

Cited by 110 publications

(60 citation statements)

References 63 publications

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“…Our findings thus invoke a view of an expanded role of the overall fold in RNA function. They also have broad implications for applications that seek out RNA as a drug target (23), use ligand binding distal to the active site of an RNA, or engineer allosteric RNA enzymes (24).…”

Section: Discussionmentioning

confidence: 99%

Single-molecule enzymology of RNA: Essential functional groups impact catalysis from a distance

Rueda

Bokinsky

Rhodes

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

138

200

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The hairpin ribozyme is a minimalist paradigm for studying RNA folding and function. In this enzyme, two domains dock by induced fit to form a catalytic core that mediates a specific backbone cleavage reaction. Here, we have fully dissected its reversible reaction pathway, which comprises two structural transitions (docking͞undocking) and a chemistry step (cleavage͞ligation), by applying a combination of single-molecule fluorescence resonance energy transfer (FRET) assays, ensemble cleavage assays, and kinetic simulations. This has allowed us to quantify the effects that modifications of essential functional groups remote from the site of catalysis have on the individual rate constants. We find that all ribozyme variants show similar fractionations into effectively noninterchanging molecule subpopulations of distinct undocking rate constants. This leads to heterogeneous cleavage activity as commonly observed for RNA enzymes. A modification at the domain junction additionally leads to heterogeneous docking. Surprisingly, most modifications not only affect docking͞undocking but also significantly impact the internal chemistry rate constants over a substantial distance from the site of catalysis. We propose that a network of coupled molecular motions connects distant parts of the RNA with its reaction site, which suggests a previously undescribed analogy between RNA and protein enzymes. Our findings also have broad implications for applications such as the action of drugs and ligands distal to the active site or the engineering of allostery into RNA. R NA enzymes (ribozymes) have been recognized as ideal model systems for studying the relationship of structure and function in RNA, because their catalytic activity directly reports the extent of native structure formation (1-3). This provides the basis for powerful modification-interference experiments in which the activity of site-specifically modified ribozymes is compared to the unmodified WT to map functionally important residues of the catalytic core. Such chemical modifications, however, may impact ribozyme function through reaction chemistry, structure formation, or both. Distinguishing these mechanisms has long been an experimental challenge. A typical example is the hairpin ribozyme, derived from the self-cleaving 359-nt negative strand of the tobacco ringspot virus satellite RNA, a member of a family of plant pathogens (4-6). A wealth of modification-interference experiments has helped to define the residues important for function of the minimal two-way junction form of this catalytic RNA, designed as the sequence with highest enzymatic activity in external substrate cleavage (4,5,7,8). Many functional groups of the 24 non-Watson-Crick base-paired nucleotides in the two internal loops of domains A and B were shown to be essential for catalytic activity (Fig. 1A). However, recent crystallographic and biochemical experiments have suggested that only two nucleobases in the ribozyme, G8 and A38, are directly involved in reaction chemistry (Fig. 1 A) (9-12). An...

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Section: Discussionmentioning

confidence: 99%

Single-molecule enzymology of RNA: Essential functional groups impact catalysis from a distance

Rueda

Bokinsky

Rhodes

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

138

200

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“…Aptamers have been conjugated to other aptamers forming " allosteric aptamers" or to ribozymes forming " aptazymes" (Soukup, 2004;Silverman, 2003). Stojanovic and Kolpashchikov first reported the design of novel allosteric sensing systems produced by coupling the MG-binding RNA aptamer through a " communication module" to each of a series of specific aptamers for ATP, FMN (flavin monomucleotide), and theophylline.…”

Section: Allosteric Aptamer-based Sensorsmentioning

confidence: 99%

New Ideas for in Vivo Detection of RNA

Novikova¹,

Afonin²,

Leontis³

2010

Biosensors

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“…It should be noted here that the ribozyme's native repertoire of ligands can be greatly expanded by fusion with an engineered aptamer, thereby constructing an allosteric ribozyme. Such biosensor modules can respond to a wide variety of signals (peptides, small organic molecules, oligonucleotides, metal ions and pH), and as a result, the allosteric ribozymes have equally wide variety of uses (158)(159)(160).…”

Section: Epiloguementioning

confidence: 99%

Insights into functional modulation of catalytic RNA activity

et al. 2008

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SummaryRNA molecules play critical roles in cell biology, and novel findings continuously broaden their functional repertoires. Apart from their well-documented participation in protein synthesis, it is now apparent that several noncoding RNAs (i.e., micro-RNAs and riboswitches) also participate in the regulation of gene expression. The discovery of catalytic RNAs had profound implications on our views concerning the evolution of life on our planet at a molecular level. A characteristic attribute of RNA, probably traced back to its ancestral origin, is the ability to interact with and be modulated by several ions and molecules of different sizes. The inhibition of ribosome activity by antibiotics has been extensively used as a therapeutical approach, while activation and substrate-specificity alteration have the potential to enhance the versatility of ribozyme-based tools in translational research. In this review, we will describe some representative examples of such modulators to illustrate the potential of catalytic RNAs as tools and targets in research and clinical approaches.

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Rube Goldberg goes (ribo)nuclear? Molecular switches and sensors made from RNA

Cited by 110 publications

References 63 publications

Single-molecule enzymology of RNA: Essential functional groups impact catalysis from a distance

Single-molecule enzymology of RNA: Essential functional groups impact catalysis from a distance

New Ideas for in Vivo Detection of RNA

Insights into functional modulation of catalytic RNA activity

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