Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution
In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5'-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA.
|Author:||Jörg Rinnenthal, Birgit Klinkert, Franz Narberhaus, Harald Schwalbe|
|Date of Publication (online):||11.11.2010|
|Year of first Publication:||2010|
|Publishing Institution:||Univ.-Bibliothek Frankfurt am Main|
|Source:||Nucleic acids research (2010) 38 (11): 3834-3847 ; doi: 10.1093/nar/gkq124|
|Institutes:||Biochemie und Chemie|
|Dewey Decimal Classification:||570 Biowissenschaften; Biologie|
© The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
|Licence (German):||Veröffentlichungsvertrag für Publikationen ohne Print on Demand|