A Disconnect between High-Affinity Binding and Efficient Regulation by Antifolates and Purines in the Tetrahydrofolate Riboswitch

Trausch, J.J.; Batey, Robert T.

doi:10.1016/j.chembiol.2013.11.012

Cited by 48 publications

(54 citation statements)

References 52 publications

(85 reference statements)

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“…Surprisingly, high-affinity riboswitch ligand analogs do not always modulate transcription (3,53,54), which adds another layer of complexity to an already daunting challenge of developing antibiotics that target the riboswitch. However, two recent reports have identified small-molecule inhibitors targeting the riboflavin riboswitch that have in vivo activity against E. coli (55) and C. difficile (60).…”

Section: Discussionmentioning

confidence: 99%

De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo

Kofoed¹,

Yan²,

Katakam³

et al. 2016

J Bacteriol

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De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5=-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuXguaB-guaA deletion in vivo. In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility. IMPORTANCEDe novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus. We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.

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Section: Discussionmentioning

confidence: 99%

De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo

Kofoed¹,

Yan²,

Katakam³

et al. 2016

J Bacteriol

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“…While the tandem glycine riboswitch remains the only riboswitch system with two homologous aptamer domains that regulate a single expression platform, another riboswitch has recently been shown to bind two ligand molecules within a single-aptamer domain, tetrahydrofolate (THF) (Trausch et al 2011;Trausch and Batey 2014). In the case of the THF riboswitch, both kinetic scaffolding and thermodynamic cooperativity have been suggested as rationale for dual ligand binding.…”

Section: Discussionmentioning

confidence: 99%

“…Scaffolding can have important consequences for kinetically controlled riboswitches (Wickiser et al 2005a,b;Trausch and Batey 2014), allowing ligand binding to occur on transcriptionally relevant time scales. As some glycine riboswitches control transcription termination, they are likely to be kinetically controlled (Serganov and Patel 2012).…”

Section: Discussionmentioning

confidence: 99%

Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

Ruff

Strobel

2014

RNA

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The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform.

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“…For example, the three-way junction (3WJ) site of the THF riboswitch [36] and the preQ 1 -II riboswitch [37] recognizes guanine through a U-purine-C base triple (Figure 2A, B). The second site in the THF riboswitch, at the pseudoknot (PK), has a slightly different arrangement of pyrimidine residues around the purine base, but still uses a uridine base to recognize the N9/N3 face (note that the N9/N3 face corresponds to the “sugar edge” of purine nucleotides) (Figure 2C) [36]. The only deviation from this theme is found in the preQ 1 -I riboswitch where the N9/N3 face of the guanine base interacts with the Watson-Crick face of the base of an adenosine residue (Figure 2D) [7-9].…”

Section: Purine Nucleobase Recognitionmentioning

confidence: 99%

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

Porter¹,

Marcano-Velázquez²,

Batey³

2014

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Over the past decade the purine riboswitch, and in particular its nucleobase-binding aptamer domain, has emerged as an important model system for exploring various aspects of RNA structure and function. Its relatively small size, structural simplicity and readily observable activity enable application of a wide variety of experimental approaches towards the study of this RNA. These analyses have yielded important insights into small molecule recognition, co-transcriptional folding and secondary structural switching, and conformational dynamics that serve as a paradigm for other RNAs. In this article, the current state of understanding of the purine riboswitch family is examined and how this growing knowledge base is starting to be exploited in the creation of novel RNA devices.

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A Disconnect between High-Affinity Binding and Efficient Regulation by Antifolates and Purines in the Tetrahydrofolate Riboswitch

Cited by 48 publications

References 52 publications

De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo

De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo

Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

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

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