Conformational dynamics of the tetracycline-binding aptamer

Förster, Ute; Weigand, Julia E.; Trojanowski, P.; Suess, Beatrix; Wachtveitl, Josef

doi:10.1093/nar/gkr835

Cited by 54 publications

(64 citation statements)

References 39 publications

(77 reference statements)

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“…Our results are consistent with a recent study by Forster et al. that explored the importance of the kinetics of ligand binding in engineered tetracycline riboswitches (33). …”

Section: Discussionmentioning

confidence: 99%

A family of synthetic riboswitches adopts a kinetic trapping mechanism

Mishler¹,

Gallivan²

2014

Nucleic Acids Research

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Riboswitches are sequences of RNA that control gene expression via RNA–ligand interactions, without the need for accessory proteins. Riboswitches consist of an aptamer that recognizes the ligand and an expression platform that couples ligand binding to a change in gene expression. Using in vitro selection, it is possible to screen large (∼1013 members) libraries of RNA sequences to discover new aptamers. However, limitations in bacterial transformation efficiency make screening such large libraries for riboswitch function in intact cells impractical. Here we show that synthetic riboswitches function in an E. coli S30 extract in a manner similar to how they function in intact E. coli cells. We discovered that, although this family of riboswitches regulates the initiation of protein translation, the fate of whether an RNA message is translated is determined during transcription. Thus, ligand binding does not bias a population of rapidly equilibrating RNA structures, but rather, co-transcriptional ligand binding kinetically traps the RNA in a conformation that supports efficient translation. In addition to providing new insights into the mechanisms of action of a family of synthetic riboswitches, our experiments suggest that it may be possible to perform selections for novel synthetic riboswitches in an in vitro system.

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“…Our results are consistent with a recent study by Forster et al. that explored the importance of the kinetics of ligand binding in engineered tetracycline riboswitches (33). …”

Section: Discussionmentioning

confidence: 99%

A family of synthetic riboswitches adopts a kinetic trapping mechanism

Mishler¹,

Gallivan²

2014

Nucleic Acids Research

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“…Nucleic acids, especially RNAs, have diverse functions in living organisms that frequently involve conformational rearrangements during ligand binding and signaling, cotranscriptional folding, catalysis in ribozymes, and the assembly of ribonucleoproteins (Al-Hashimi and Walter 2008;Nick Taylor et al 2008;Forster et al 2012). Molecules that interfere with the activity of any of these RNAs are potential drugs, particularly if they can distinguish between eukaryotic and prokaryotic RNAs.…”

Section: Introductionmentioning

confidence: 99%

An adaptable pentaloop defines a robust neomycin-B RNA aptamer with conditional ligand-bound structures

İlgü

Fulton²,

Yennamalli

et al. 2014

RNA

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Aptamers can be highly specific for their targets, which implies precise molecular recognition between aptamer and target. However, as small polymers, their structures are more subject to environmental conditions than the more constrained longer RNAs such as those that constitute the ribosome. To understand the balance between structural and environmental factors in establishing ligand specificity of aptamers, we examined the RNA aptamer (NEO1A) previously reported as specific for neomycin-B. We show that NEO1A can recognize other aminoglycosides with similar affinities as for neomycin-B and its aminoglycoside specificity is strongly influenced by ionic strength and buffer composition. NMR and 2-aminopurine (2AP) fluorescence studies of the aptamer identified a flexible pentaloop and a stable binding pocket. Consistent with a wellstructured binding pocket, docking analysis results correlated with experimental measures of the binding energy for most ligands. Steady state fluorescence studies of 2AP-substituted aptamers confirmed that A16 moves to a more solvent accessible position upon ligand binding while A14 moves to a less solvent accessible position, which is most likely a base stack. Analysis of binding affinities of NEO1A sequence variants showed that the base in position 16 interacts differently with each ligand and the interaction is a function of the buffer constituents. Our results show that the pentaloop provides NEO1A with the ability to adapt to external influences on its structure, with the critical base at position 16 adjusting to incorporate each ligand into a stable pocket by hydrophobic interactions and/or hydrogen bonds depending on the ligand and the ionic environment.

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“…30,36 The QT-1 and QT-2 sequences exhibit similar changes in the CD spectra, which demonstrates that these sequences may bind L-Arm in a manner similar to that found for the WT aptamer. Conversely, for many randomly selected genotype neighborhoods and previously reported clone 12-28 mutants, 19 no changes in the CD signal were found in the presence of L-Arm, which is consistent with their extremely low ligand-binding affinity.…”

Section: Cc-by-nc-nd 40 International Licensementioning

confidence: 75%

Exploring the mutational robustness of nucleic acids by searching genotype neighbourhoods in sequence space

Zhou

Sun

Xia

et al. 2016

Preprint

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To assess the mutational robustness of nucleic acids, many genome- and protein-level studies have been performed; in these investigations, nucleic acids are treated as genetic information carriers and transferrers. However, the molecular mechanism through which mutations alter the structural, dynamic and functional properties of nucleic acids is poorly understood. Here, we performed SELEX in silico study to investigate the fitness distribution of the nucleic acid genotype neighborhood in a sequence space for L-Arm binding aptamer. Although most mutants of the L-Arm-binding aptamer failed to retain their ligand-binding ability, two novel functional genotype neighborhoods were isolated by SELEX in silico and experimentally verified to have similar binding affinity (Kd = 69.3 μM and 110.7 μM) as the wild-type aptamer (Kd = 114.4 μM). Based on data from the current study and previous research, mutational robustness is strongly influenced by the local base environment and ligand-binding mode, whereas bases distant from the binding pocket provide potential evolutionary pathways to approach global fitness maximum. Our work provides an example of successful application of SELEX in silico to optimize an aptamer and demonstrates the strong sensitivity of mutational robustness to the site of genetic variation.

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Conformational dynamics of the tetracycline-binding aptamer

Cited by 54 publications

References 39 publications

A family of synthetic riboswitches adopts a kinetic trapping mechanism

A family of synthetic riboswitches adopts a kinetic trapping mechanism

An adaptable pentaloop defines a robust neomycin-B RNA aptamer with conditional ligand-bound structures

Exploring the mutational robustness of nucleic acids by searching genotype neighbourhoods in sequence space

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