Development and Application of a High-Throughput Assay for glmS Riboswitch Activators

Blount, Ken; Puskarz, Izabela; Penchovsky, Robert; Breaker, Ronald R.

doi:10.4161/rna.3.2.3102

Cited by 69 publications

(64 citation statements)

References 23 publications

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“…Therefore, it would be difficult to protonate the phosphate group of GlcN6P, which interacts with a bridging Mg 2+ in the binding site of glmS that is mainly responsible for the binding of GlcN6P and stability of the complex. Hence, the activity of glmS in the presence of GlcN6P decreases with decreasing concentration of Mg 2+ , and GlcN, which lacks the phosphate group on position six, is a poorer activator of glmS than GlcN6P due to the fact that it forms a less stable complex with glmS McCarthy et al 2005;Blount et al 2006;Jansen et al 2006;Link et al 2006;Mayer and Famulok 2006). Furthermore, the fact that the substrate analog, Glc6P, which has an hydroxyl functional group instead of the amine group, binds to glmS in a pH-independent fashion (Klein et al 2007b), suggests that the amine group and not the phosphate group is responsible for the pH-dependent effect of GlcN6P binding.…”

Section: Implications Of the Pk A Shift Of The Amine Group Of Glcn6p mentioning

confidence: 99%

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Yao¹,

Hamelberg²

2010

RNA

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The GlmS ribozyme is believed to exploit a general acid-base catalytic mechanism in the presence of glucosamine-6-phosphate (GlcN6P) to accelerate self-cleavage by approximately six orders of magnitude. The general acid and general base are not known, and the role of the GlcN6P cofactor is even less well understood. The amine group of GlcN6P has the ability to either accept or donate a proton and could therefore potentially act as an acid or a base. In order to decipher the role of GlcN6P in the self-cleavage of glmS, we have determined the preferred protonation state of the amine group in the wild-type and an inactive G40A mutant using molecular dynamics simulations and free energy calculations. Here we show that, upon binding of GlcN6P to wild-type glmS, the pK a of the amine moiety is altered by the active site environment, decreasing by about 2.2 from a solution pK a of about 8.2. On the other hand, we show that the pK a of the amine group slightly increases to about 8.4 upon binding to the G40A inactive mutant of glmS. These results suggest that GlcN6P acts as a general acid in the self-cleavage of glmS. Upon binding to glmS, GlcN6P can easily release a proton to the 59-oxygen of G1 during self-cleavage of the backbone phosphodiester bond. However, in the G40A inactive mutant of glmS, the results suggest that the ability of GlcN6P to easily release its proton is diminished, in addition to the possible lack of G40 as an effective base.

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Section: Implications Of the Pk A Shift Of The Amine Group Of Glcn6p mentioning

confidence: 99%

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Yao¹,

Hamelberg²

2010

RNA

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“…Blount and coworkers also developed a HTS assay for glmS ribozyme cleavage based on FRET [80]. To validate the screening assay, a focused library of GlcN6P analogues whose affinities for the ribozymes were determined by conventional radiolabeled method was used.…”

Section: Riboswitchesmentioning

confidence: 99%

Functional Nucleic Acids in High Throughput Screening and Drug Discovery

Srivatsan¹,

Famulok²

2007

CCHTS

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In vitro selection can be used to generate functional nucleic acids such as aptamers and ribozymes that can recognize a variety of molecules with high affinity and specificity. Most often these recognition events are associated with structural alterations that can be converted into detectable signals. Several signaling aptamers and ribozymes constructed by both design and selection have been successfully utilized as sensitive detection reagents. Here we summarize the development of different types of signaling nucleic acids, and approaches that have been implemented in the screening format.

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“…Three of these riboswitch classes have been revealed as important cellular targets of antibacterial small molecules whose mechanism of action had not been previously defined (9)(10)(11)(12)(13). More recently, several publications have demonstrated that novel small molecules that bind to selected riboswitch aptamers with affinities comparable to that of the cognate ligand can be rationally identified and optimized (14)(15)(16)(17)(18)(19)(20)(21).…”

mentioning

confidence: 99%

Novel Riboswitch-Binding Flavin Analog That Protects Mice against Clostridium difficile Infection without Inhibiting Cecal Flora

Blount

Megyola

Plummer

et al. 2015

Antimicrob Agents Chemother

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Despite nearly 85 years of successful antibacterial chemotherapy, bacterial infections still pose a serious threat to human health. Continually emerging drug-resistant bacteria make existing agents less effective, and a paucity of new agents and decreased development effort from the pharmaceutical industry provide little hope of replenishing the arsenal (1, 2). Complications and mortality due to bacterial infections continue to increase, already reaching epidemic proportions in some areas of the world. If humans are to regain the upper hand in fighting bacterial infections, then innovation, investment, and new antibacterial agents will be needed. Although some of the barriers to the discovery and approval of new compounds are economic or policy related, there is also a desperate need for new targets and new mechanisms of action. A renewed effort to expand our knowledge of bacterial physiology and to translate discoveries into the clinic will be needed to address these challenges and to reinvigorate antibiotic development pipelines.Recent advances in our understanding of how bacteria maintain physiological homeostasis revealed a promising class of potential antibiotic targets called riboswitches-noncoding mRNAs that form a structured receptor (or aptamer) which can directly bind to a specific small-molecule ligand or ion and thereby regulate gene expression (3-5). Ligand binding to a riboswitch aptamer stabilizes a conformationally distinct architecture in the mRNA that modulates the expression of the adjacent coding region(s) (4-8). To date, more than 35 riboswitch classes have been discovered and characterized (7). Three of these riboswitch classes have been revealed as important cellular targets of antibacterial small molecules whose mechanism of action had not been previously defined (9-13). More recently, several publications have demonstrated that novel small molecules that bind to selected riboswitch aptamers with affinities comparable to that of the cognate ligand can be rationally identified and optimized (14-21).In some cases, synthetic or natural riboswitch ligand analogs have demonstrated potent antibacterial activity (12-15, 20, 22). For example, the phosphorylated form of roseoflavin (RoF) (

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Development and Application of a High-Throughput Assay for glmS Riboswitch Activators

Cited by 69 publications

References 23 publications

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Functional Nucleic Acids in High Throughput Screening and Drug Discovery

Novel Riboswitch-Binding Flavin Analog That Protects Mice against Clostridium difficile Infection without Inhibiting Cecal Flora

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