TU Darmstadt / ULB / TUbiblio

Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity.

Weigand, J. E. and Schmidtke, S. R. and Will, T. J. and Duchardt-Ferner, E. and Hammann, Christian and Wöhnert, J. and Suess, Beatrix (2011):
Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity.
In: Nucleic acids research, pp. 3363-3372, 39, (8), ISSN 1362-4962, [Article]

Abstract

While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch.

Item Type: Article
Erschienen: 2011
Creators: Weigand, J. E. and Schmidtke, S. R. and Will, T. J. and Duchardt-Ferner, E. and Hammann, Christian and Wöhnert, J. and Suess, Beatrix
Title: Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity.
Language: English
Abstract:

While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch.

Journal or Publication Title: Nucleic acids research
Volume: 39
Number: 8
Divisions: 10 Department of Biology > Ribogenetics
10 Department of Biology > Synthetic Genetic Circuits
?? fb10_mikrobiologie ??
10 Department of Biology
Date Deposited: 25 Jan 2011 07:21
Export:

Optionen (nur für Redakteure)

View Item View Item