Mechanistic insights into temperature-dependent regulation of the simple cyanobacterial hsp17 RNA thermometer at base-pair resolution

Abstract

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 hsp17rep 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 hsp17rep. 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.

Structural Insights into Conformation Differences between DNA/TNA and RNA/TNA Chimeric Duplexes

“…[14] As expected, k ex (C2-G19) increased with temperature until reaching a detection limit (asymptote) at 25 °C. By comparison, exchange rates of other base pairs followed a much slower temperature-dependent exponential curve, reaching their detection limits at ≥40 °C.…”

“…Higher values at the ends for ΔH Diss (GC) than ΔH Diss (AT) in the middle of the sequence likely reflect the formation of all three hydrogen bonds between guanine and cytosine bases. Compared to ΔG Diss reported for central bases in DNA duplexes (24 kJ/mol to 32.6 kJ/mol) [14b, 15a] ΔG Diss for DNA/TNA central pairs were mostly at the lower end of the range. The asymmetry of duplex breathing was reflected with a distribution of ΔG Diss values.…”

Structure and mechanism of a molecular rheostat, an RNA thermometer that modulates immune evasion byNeisseria meningitidis

“…The architecture of the CssA thermometer is reminiscent of the cyanobacterial hsp17 thermometer (18). In the cyanobacterial structure (one of the smallest thermometers) the 3 nucleotide bulge separates the start codon from the RBS.…”

“…To date, there are remarkably few reports of the structure of RNA thermometers (17,18). The Cyanobacterial thermometer, one of the smallest naturally occurring RNA thermometers (18), consists of only 24 nucleotides, while a 34 nucleotide microdomain of a larger RNA structure has also been characterized (17).…”

“…To date, there are remarkably few reports of the structure of RNA thermometers (17,18). The Cyanobacterial thermometer, one of the smallest naturally occurring RNA thermometers (18), consists of only 24 nucleotides, while a 34 nucleotide microdomain of a larger RNA structure has also been characterized (17). The cssA RNA thermometer usually contains two copies of an 8 nucleotide sequence (TATACTTA) located just upstream of the RBS and 15 nucleotides upstream of the cssA start codon (Figure 1) (7,19); with this 8 nucleotide repeat, the sequence of the 5′-UTR is remarkably conserved.…”