Mechanistic insights into temperature-dependent regulation of the simple cyanobacterial hsp17 RNA thermometer at base-pair resolution.
The cyanobacterial hsp17 ribonucleicacid thermometer (RNAT) is one of the smallest naturally occurring RNAT. It forms a single hairpin with an internal 1×3-bulge separating the start codon in stem I from the ribosome binding site (RBS) in stem II. We investigated the temperature-dependent regulation of hsp17 by mapping individual base-pair stabilities from solvent exchange nuclear magnetic resonance (NMR) spectroscopy. The wild-type RNAT was found to be stabilized by two critical CG base pairs (C14-G27 and C13-G28). Replacing the internal 1×3 bulge by a stable CG base pair in hsp17(rep) significantly increased the global stability and unfolding cooperativity as evidenced by circular dichroism spectroscopy. From the NMR analysis, remote stabilization and non-nearest neighbour effects exist at the base-pair level, in particular for nucleotide G28 (five nucleotides apart from the side of mutation). Individual base-pair stabilities are coupled to the stability of the entire thermometer within both the natural and the stabilized RNATs by enthalpy-entropy compensation presumably mediated by the hydration shell. At the melting point the Gibbs energies of the individual nucleobases are equalized suggesting a consecutive zipper-type unfolding mechanism of the RBS leading to a dimmer-like function of hsp17 and switch-like regulation behaviour of hsp17(rep). The data show how minor changes in the nucleotide sequence not only offset the melting temperature but also alter the mode of temperature sensing. The cyanobacterial thermosensor demonstrates the remarkable adjustment of natural RNATs to execute precise temperature control.
Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 7, D-60438 Frankfurt/Main, Germany.
Dominic
D
Wagner
Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 7, D-60438 Frankfurt/Main, Germany.
Jörg
J
Rinnenthal
Microbial Biology, Ruhr University, Universitätsstr. 150, D-44780 Bochum, Germany.
Franz
F
Narberhaus
Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 7, D-60438 Frankfurt/Main, Germany Schwalbe@nmr.uni-frankfurt.de.
Harald
http://orcid.org/0000-0001-5693-7909
H
Schwalbe
D001426:Bacterial Proteins / Q000235:genetics*; D020029:Base Pairing; D019277:Entropy; D006360:Heat-Shock Proteins / Q000235:genetics*; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D059370:RNA Folding; D012329:RNA, Bacterial / Q000737:chemistry*; D046939:Synechocystis / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D011522:Protons; D012329:RNA, Bacterial; D014867:Water