The purine riboswitch as a model system for exploring RNA biology and chemistry

Porter, Ely; Marcano-Velázquez, Joan G.; Batey, Robert T.

doi:10.1016/j.bbagrm.2014.02.014

Cited by 31 publications

(48 citation statements)

References 130 publications

(213 reference statements)

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“…This junction comprises up to five nucleotides for the U,C,C group vs. only two for guanine and adenine riboswitches. Additionally, nucleotide position 47 is highly conserved as a U residue in guanine riboswitches and is predicted to closely approach the ligand in atomic-resolution structures (26,27,31). However, this position is not well conserved in U,C,C-group RNAs.…”

Section: Resultsmentioning

confidence: 99%

Bioinformatic analysis of riboswitch structures uncovers variant classes with altered ligand specificity

Weinberg

Nelson

Lünse

et al. 2017

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

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Riboswitches are RNAs that form complex, folded structures that selectively bind small molecules or ions. As with certain groups of protein enzymes and receptors, some riboswitch classes have evolved to change their ligand specificity. We developed a procedure to systematically analyze known riboswitch classes to find additional variants that have altered their ligand specificity. This approach uses multiple-sequence alignments, atomic-resolution structural information, and riboswitch gene associations. Among the discoveries are unique variants of the guanine riboswitch class that most tightly bind the nucleoside 2′-deoxyguanosine. In addition, we identified variants of the glycine riboswitch class that no longer recognize this amino acid, additional members of a rare flavin mononucleotide (FMN) variant class, and also variants of c-di-GMP-I and -II riboswitches that might recognize different bacterial signaling molecules. These findings further reveal the diverse molecular sensing capabilities of RNA, which highlights the potential for discovering a large number of additional natural riboswitch classes.iboswitches are structured noncoding RNA domains that regulate gene expression in response to the selective binding of small-molecule or ion ligands. The discovery of numerous classes of riboswitches has helped reveal how RNAs can form exquisitely precise ligand-binding pockets using only the four common RNA nucleotides (1-4). Furthermore, each discovery links the riboswitch ligand to the protein products of the genes under regulation. Recent riboswitch findings have exposed unique facets of biology, such as the widespread molecular mechanisms that confer fluoride (5) or guanidine (6) resistance, that maintain metal ion homeostasis (7-9), and that control important bacterial processes such as sporulation, biofilm formation, and chemotaxis (10-14). Thus, the identification of additional riboswitch classes promises to offer insights into otherwise hidden biological processes and their regulation.Riboswitch variants have been reported previously, wherein the ligand-binding "aptamer" domain has mutated to accommodate a different metabolite or signaling compound. The identification of such RNAs provides rare opportunities to study how small changes in RNA sequence can lead to major changes in smallmolecule ligand affinity. There have been seven examples, either experimentally validated or suspected, of ligand specificity changes reported to date. These include guanine aptamer variations present in riboswitches for adenine (15) and 2′-deoxyguanosine (2′-dG) (16), c-di-GMP-I aptamer variations that result in riboswitches that bind the recently discovered bacterial signaling molecule c-AMP-GMP (13, 14), and coenzyme B 12 aptamer changes (17, 18) that yield riboswitches selective for aquocobalamin (19). Three additional ligand specificity changes are suspected. Namely, some molybdenum cofactor riboswitches appear to exploit an altered aptamer structure to selectively recognize tungsten cofactor (20), certain flavin mononu...

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Section: Resultsmentioning

confidence: 99%

Bioinformatic analysis of riboswitch structures uncovers variant classes with altered ligand specificity

Weinberg

Nelson

Lünse

et al. 2017

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

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“…Over the past decade, isolated riboswitch aptamer domains have been studied extensively using a broad spectrum of in vitro approaches (1). This study has taken the next step by investigating effector-dependent regulation of the B. subtilis pbuE adenine-responsive riboswitch (which contains one of the best characterized aptamers) in the context of transcription and under cellular conditions that more closely match the speed of RNA transcription, salt concentrations, and molecular crowdedness of the native environment of the riboswitch.…”

Section: Discussionmentioning

confidence: 99%

“…The folding and binding properties of the pbuE aptamer domain, along with several other representative purine riboswitch aptamers, have been studied extensively using a broad set of structural and biophysical techniques (1). Notably, the first comprehensive analysis of co-transcriptional folding of an RNA by single-molecule techniques was recently reported by Frieda and Block (3) using the pbuE riboswitch.…”

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confidence: 99%

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Structure-guided Mutational Analysis of Gene Regulation by the Bacillus subtilis pbuE Adenine-responsive Riboswitch in a Cellular Context

Marcano-Velázquez

Batey

2015

Journal of Biological Chemistry

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Background: Riboswitches regulate purine biosynthesis and transport in many bacteria. Results: Mutagenic analysis of an adenine-responsive riboswitch revealed features important for efficient co-transcriptional regulation. Conclusion: Adenine binding to the riboswitch results in a local barrier to strand exchange by the transcriptional terminator, whose formation is facilitated by a stem-loop element. Significance: This is the first study comprehensively analyzing an RNA structural switch in a cellular context.Riboswitches are a broadly distributed form of RNA-based gene regulation in Bacteria and, more rarely, Archaea and Eukarya. Most often found in the 5-leader sequence of bacterial mRNAs, they are generally composed of two functional domains: a receptor (aptamer) domain that binds an effector molecule and a regulatory domain (or expression platform) that instructs the expression machinery. One of the most studied riboswitches is the Bacillus subtilis adenine-responsive pbuE riboswitch, which regulates gene expression at the transcriptional level, up-regulating expression in response to increased intracellular effector concentrations. In this work, we analyzed sequence and structural elements that contribute to efficient ligand-dependent regulatory activity in a co-transcriptional and cellular context. Unexpectedly, we found that the P1 helix, which acts as the antitermination element of the switch in this RNA, supported ligand-dependent activation of a reporter gene over a broad spectrum of lengths from 3 to 10 bp. This same trend was also observed using a minimal in vitro single-turnover transcription assay, revealing that this behavior is intrinsic to the RNA sequence. We also found that the sequences at the distal tip of the terminator not directly involved in alternative secondary structure formation are highly important for efficient regulation. These data strongly support a model in which the switch is highly localized to the P1 helix adjacent to the ligandbinding pocket that likely presents a local kinetic block to invasion of the aptamer by the terminator.Since their discovery and structural characterization, purineresponsive riboswitches have emerged as an important model system for exploring small molecule-RNA interactions, RNA structure, in vitro and in vivo folding, and conformational dynamics (reviewed in Ref. 1). Purine riboswitches present themselves as an ideal model system to explore various aspects of RNA chemistry and biology because of their small size and simple architecture of the ligand-binding domain, very well behaved folding properties, and multiple activities (small-molecule recognition and regulation of gene expression). However, the majority of studies of riboswitches have focused solely upon the ligand-binding (aptamer) domain in isolation using in vitro biochemical, biophysical and structural approaches. Thus, the wealth of information about the structure and function of the aptamer domain is poorly linked to an understanding of regulatory activity, as is the case for most clas...

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“…[10,11,27,28]). In contrast, the unbound state is by far less well characterized, especially at the atomistic level.…”

Section: Introductionmentioning

confidence: 99%

Ligand-mediated and tertiary interactions cooperatively stabilize the P1 region in the guanine-sensing riboswitch

Hanke

Gohlke

2017

PLoS ONE

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Riboswitches are genetic regulatory elements that control gene expression depending on ligand binding. The guanine-sensing riboswitch (Gsw) binds ligands at a three-way junction formed by paired regions P1, P2, and P3. Loops L2 and L3 cap the P2 and P3 helices and form tertiary interactions. Part of P1 belongs to the switching sequence dictating the fate of the mRNA. Previous studies revealed an intricate relationship between ligand binding and presence of the tertiary interactions, and between ligand binding and influence on the P1 region. However, no information is available on the interplay among these three main regions in Gsw. Here we show that stabilization of the L2-L3 region by tertiary interactions, and the ligand binding site by ligand binding, cooperatively influences the structural stability of terminal base pairs in the P1 region in the presence of Mg2+ ions. The results are based on molecular dynamics simulations with an aggregate simulation time of ~10 μs across multiple systems of the unbound state of the Gsw aptamer and a G37A/C61U mutant, and rigidity analyses. The results could explain why the three-way junction is a central structural element also in other riboswitches and how the cooperative effect could become contextual with respect to intracellular Mg2+ concentration. The results suggest that the transmission of allosteric information to P1 can be entropy-dominated.

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The purine riboswitch as a model system for exploring RNA biology and chemistry

Cited by 31 publications

References 130 publications

Bioinformatic analysis of riboswitch structures uncovers variant classes with altered ligand specificity

Bioinformatic analysis of riboswitch structures uncovers variant classes with altered ligand specificity

Structure-guided Mutational Analysis of Gene Regulation by the Bacillus subtilis pbuE Adenine-responsive Riboswitch in a Cellular Context

Ligand-mediated and tertiary interactions cooperatively stabilize the P1 region in the guanine-sensing riboswitch

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