Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch

Weigand, Julia E.; Gottstein‐Schmidtke, Sina R.; Demolli, Shemsi; Groher, Florian; Duchardt‐Ferner, Elke; Wöhnert, Jens; Suess, Beatrix

doi:10.1002/cbic.201402067

Cited by 14 publications

(14 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, depriving A17 of the Hoogsteen edge hydrogen acceptors should lead to breaking of the G5–A17 contact and diminishing riboswitch activity. Indeed, the A17C riboswitch mutant shows reduced gene regulatory activity against NEO ( 50 ).…”

Section: Resultsmentioning

confidence: 99%

Molecular mechanisms for dynamic regulation of N1 riboswitch by aminoglycosides

Kulik

Mori

Sugita

et al. 2018

Nucleic Acids Research

View full text Add to dashboard Cite

A synthetic riboswitch N1, inserted into the 5′-untranslated mRNA region of yeast, regulates gene expression upon binding ribostamycin and neomycin. Interestingly, a similar aminoglycoside, paromomycin, differing from neomycin by only one substituent (amino versus hydroxyl), also binds to the N1 riboswitch, but without affecting gene expression, despite NMR evidence that the N1 riboswitch binds all aminoglycosides in a similar way. Here, to explore the details of structural dynamics of the aminoglycoside-N1 riboswitch complexes, we applied all-atom molecular dynamics (MD) and temperature replica-exchange MD simulations in explicit solvent. Indeed, we found that ribostamycin and neomycin affect riboswitch dynamics similarly but paromomycin allows for more flexibility because its complex lacks the contact between the distinctive 6′ hydroxyl group and the G9 phosphate. Instead, a transient hydrogen bond of 6′-OH with A17 is formed, which partially diminishes interactions between the bulge and apical loop of the riboswitch, likely contributing to riboswitch inactivity. In many ways, the paromomycin complex mimics the conformations, interactions, and Na+ distribution of the free riboswitch. The MD-derived interaction network helps understand why riboswitch activity depends on aminoglycoside type, whereas for another aminoglycoside-binding site, aminoacyl-tRNA site in 16S rRNA, activity is not discriminatory.

show abstract

Section: Resultsmentioning

confidence: 99%

Molecular mechanisms for dynamic regulation of N1 riboswitch by aminoglycosides

Kulik

Mori

Sugita

et al. 2018

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…During an in vivo screen of a neomycin riboswitch library with a randomized apical hexaloop (nt 13-18), sequences with a U to C mutation at position 14 were identified as functional despite their apparent deviation from the structurally important U-turn consensus sequence ( Fig. 1C; Weigand et al 2014). However, compared to the WT riboswitch, their regulatory power is slightly diminished.…”

Section: Resultsmentioning

confidence: 99%

“…In a recent screen aimed at identifying functional sequence variants of the apical loop of the neomycin riboswitch, we found that a replacement of U14 by a C residue ( Fig. 1A) also results in functional riboswitches (Weigand et al 2014). Such a mutation should interfere with the proper folding and the stability of the U-turn motif because C lacks an imino proton and is thus not able to form a hydrogen bond analogous to the one between the U imino group and the phosphate backbone in the canonical U-turn motif.…”

Section: Introductionmentioning

confidence: 99%

Building a stable RNA U-turn with a protonated cytidine

Gottstein‐Schmidtke

Duchardt‐Ferner

Groher

et al. 2014

RNA

Self Cite

View full text Add to dashboard Cite

The U-turn is a classical three-dimensional RNA folding motif first identified in the anticodon and T-loops of tRNAs. It also occurs frequently as a building block in other functional RNA structures in many different sequence and structural contexts. U-turns induce sharp changes in the direction of the RNA backbone and often conform to the 3-nt consensus sequence 5 ′ -UNR-3 ′ (N = any nucleotide, R = purine). The canonical U-turn motif is stabilized by a hydrogen bond between the N3 imino group of the U residue and the 3 ′ phosphate group of the R residue as well as a hydrogen bond between the 2 ′ -hydroxyl group of the uridine and the N7 nitrogen of the R residue. Here, we demonstrate that a protonated cytidine can functionally and structurally replace the uridine at the first position of the canonical U-turn motif in the apical loop of the neomycin riboswitch. Using NMR spectroscopy, we directly show that the N3 imino group of the protonated cytidine forms a hydrogen bond with the backbone phosphate 3′ from the third nucleotide of the U-turn analogously to the imino group of the uridine in the canonical motif. In addition, we compare the stability of the hydrogen bonds in the mutant U-turn motif to the wild type and describe the NMR signature of the C+-phosphate interaction. Our results have implications for the prediction of RNA structural motifs and suggest simple approaches for the experimental identification of hydrogen bonds between protonated C-imino groups and the phosphate backbone.

show abstract

“…Subsequently 20 injections of 2.0 μl at a 180 s interval were made. The sample cell was stirred with a speed of 750 rpm (60). The software NITPIC was used to integrate the injection peaks to yield the associated heat for each injection (61,62), the experimental binding isotherm was subsequently plotted and the curve fit with a one-site binding model using the software SEDPHAT (63).…”

Section: Methodsmentioning

confidence: 99%

Structure guided fluorescence labeling reveals a two-step binding mechanism of neomycin to its RNA aptamer

Gustmann

Segler

Gophane

et al. 2018

Nucleic Acids Research

View full text Add to dashboard Cite

The ability of the cytidine analog Çmf to act as a position specific reporter of RNA-dynamics was spectroscopically evaluated. Çmf-labeled single- and double-stranded RNAs differ in their fluorescence lifetimes, quantum yields and anisotropies. These observables were also influenced by the nucleobases flanking Çmf. This conformation and position specificity allowed to investigate the binding dynamics and mechanism of neomycin to its aptamer N1 by independently incorporating Çmf at four different positions within the aptamer. Remarkably fast binding kinetics of neomycin binding was observed with stopped-flow measurements, which could be satisfactorily explained with a two-step binding. Conformational selection was identified as the dominant mechanism.

show abstract

Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch

Cited by 14 publications

References 46 publications

Molecular mechanisms for dynamic regulation of N1 riboswitch by aminoglycosides

Molecular mechanisms for dynamic regulation of N1 riboswitch by aminoglycosides

Building a stable RNA U-turn with a protonated cytidine

Structure guided fluorescence labeling reveals a two-step binding mechanism of neomycin to its RNA aptamer

Contact Info

Product

Resources

About