Ligand‐Induced Folding of the Adenosine Deaminase A‐Riboswitch and Implications on Riboswitch Translational Control

Rieder, Renate; Lang, Kathrin; Graber, Dagmar; Micura, Ronald

doi:10.1002/cbic.200700057

Cited by 170 publications

(242 citation statements)

References 34 publications

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“…The add A-riboswitch was chosen as the basis for this study because it functions at the translational level via a relatively simple mechanism that could potentially operate in any bacterial system (18,19) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

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Reengineering orthogonally selective riboswitches

Dixon

Duncan²,

Geerlings³

et al. 2010

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

151

163

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The ability to independently control the expression of multiple genes by addition of distinct small-molecule modulators has many applications from synthetic biology, functional genomics, pharmaceutical target validation, through to gene therapy. Riboswitches are relatively simple, small-molecule-dependent, protein-free, mRNA genetic switches that are attractive targets for reengineering in this context. Using a combination of chemical genetics and genetic selection, we have developed riboswitches that are selective for synthetic "nonnatural" small molecules and no longer respond to the natural intracellular ligands. The orthogonal selectivity of the riboswitches is also demonstrated in vitro using isothermal titration calorimetry and x-ray crystallography. The riboswitches allow highly responsive, dose-dependent, orthogonally selective, and dynamic control of gene expression in vivo. It is possible that this approach may be further developed to reengineer other natural riboswitches for application as small-molecule responsive genetic switches in both prokaryotes and eukaryotes.

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Section: Resultsmentioning

confidence: 99%

“…1A and B). The U74 nucleobase is believed to be the initial point of ligand recognition, through Watson-Crick base pairing, after which the loop J2/3 closes around the ligand, with additional contact to the 2′-OH of U22 through the purine N7 (18,19,25,27).…”

Section: Resultsmentioning

confidence: 99%

Reengineering orthogonally selective riboswitches

Dixon

Duncan²,

Geerlings³

et al. 2010

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

151

163

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“…One obvious explanation may be that it is important to shut down lysC expression irreversibly in the presence of lysine. For instance, we have shown previously that translation regulation is performed under thermodynamic control for the add adenine riboswitch (36,37), allowing structural reversibility between both ligand-free and ligand-bound riboswitch conformations. However, in contrast to add, ligand binding to the riboswitch ensures rapid mRNA degradation and consequently definitive repression of gene expression, a function we consider to be dominant over translation inhibition.…”

Section: Discussionmentioning

confidence: 99%

Dual-acting riboswitch control of translation initiation and mRNA decay

Caron

Bastet

Lussier

et al. 2012

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

150

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Riboswitches are mRNA regulatory elements that control gene expression by altering their structure in response to specific metabolite binding. In bacteria, riboswitches consist of an aptamer that performs ligand recognition and an expression platform that regulates either transcription termination or translation initiation. Here, we describe a dual-acting riboswitch from Escherichia coli that, in addition to modulating translation initiation, also is directly involved in the control of initial mRNA decay. Upon lysine binding, the lysC riboswitch adopts a conformation that not only inhibits translation initiation but also exposes RNase E cleavage sites located in the riboswitch expression platform. However, in the absence of lysine, the riboswitch folds into an alternative conformation that simultaneously allows translation initiation and sequesters RNase E cleavage sites. Both regulatory activities can be individually inhibited, indicating that translation initiation and mRNA decay can be modulated independently using the same conformational switch. Because RNase E cleavage sites are located in the riboswitch sequence, this riboswitch provides a unique means for the riboswitch to modulate RNase E cleavage activity directly as a function of lysine. This dual inhibition is in contrast to other riboswitches, such as the thiamin pyrophosphate-sensing thiM riboswitch, which triggers mRNA decay only as a consequence of translation inhibition. The riboswitch control of RNase E cleavage activity is an example of a mechanism by which metabolite sensing is used to regulate gene expression of single genes or even large polycistronic mRNAs as a function of environmental changes.gene regulation | RNA degradosome | translational control S ince the first demonstration that translation attenuation regulates the expression of the tryptophan operon (1), accumulating evidence has revealed the importance of posttranscriptional regulation in prokaryotes and eukaryotes alike. Posttranscriptional regulators include RNA molecules that operate through several mechanisms to control a wide range of physiological responses (2). Among newly identified RNA regulators are riboswitches, which are located in untranslated regions of several mRNAs and that modulate gene expression at the level of transcription, translation, or splicing (3). Riboswitches are highly structured regulatory domains that directly sense cellular metabolites such as amino acids, carbohydrates, coenzymes, and nucleobases. These genetic switches are composed of two modular domains consisting of an aptamer and an expression platform. The aptamer is involved in the specific recognition of the metabolite, and the expression platform is used to control gene expression by altering the structure of the mRNA. Recent bioinformatic analyses have reported the existence of several new RNA motifs exhibiting unconventional expression platforms (4, 5), suggesting that riboswitches using different regulation mechanisms are still likely to be discovered (6).The lysine riboswitch was first...

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“…Studies on a C74U variant of the guanine sensing aptamer domain from B. subtilis xpt-pbuX indicate that the unbound RNA has a well defined global structure but that local gating motions at the binding pocket allow backside ligand entry that in turn induces a lid closing motion [13] (Figure 3a). In contrast, studies on the Vibrio vulnificus adenosine deaminase (add) riboswitch suggest that the ligand shifts a preexisting equilibrium between free and ligand bound conformations, leading to regulation at the translational level [14] (Figure 3b). For the structurally distinct aptamer domain of the Escherichia coli thiamine pyrophosphate (TPP) riboswitch, the RNA was singly labeled with 2-aminopurine nucleobases at various strategically chosen positions [15].…”

Section: Probing Conformational Changes In Aptamer Domainsmentioning

confidence: 98%

RNA dynamics: it is about time

Al‐Hashimi

Walter

2008

Current Opinion in Structural Biology

298

295

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SummaryMany recently discovered RNA functions rely on highly complex multi-step conformational transitions that occur in response to an array of cellular signals. These dynamics accompany and guide, for example, RNA co-transcriptional folding, ligand sensing and signaling, site-specific catalysis in ribozymes, and the hierarchically ordered assembly of ribonucleoproteins. RNA dynamics are encoded by both the inherent properties of RNA structure, spanning many motional modes with a large range of amplitudes and timescales, and external trigger factors, ranging from proteins, nucleic acids, metal ions, metabolites, and vitamins to temperature and even directional RNA biosynthesis itself. Here, we review recent advances in our understanding of RNA dynamics as highlighted by biophysical tools.

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Ligand‐Induced Folding of the Adenosine Deaminase A‐Riboswitch and Implications on Riboswitch Translational Control

Cited by 170 publications

References 34 publications

Reengineering orthogonally selective riboswitches

Reengineering orthogonally selective riboswitches

Dual-acting riboswitch control of translation initiation and mRNA decay

RNA dynamics: it is about time

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