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

D Wagner,H Schwalbe,F Narberhaus,J Rinnenthal

doi:10.1093/nar/gkv414

D Wagner, H Schwalbe + Show 2 more

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https://doi.org/10.1093/nar/gkv414

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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.

Highlights

Changes in ambient temperature affect the integrity and performance of many cellular structures and processes
The nuclear magnetic resonance (NMR) assignments of hsp17 and hsp17rep are in agreement with the secondary structure models that were previously determined by chemical and enzymatic probing at 28◦C [16]
While the shuA thermometer and other virulence-related thermosensors need to respond to a defined temperature of 37◦C, heat shock thermometers such as the ones upstream of the hsp17 or agsA genes need to modulate expression in response to wide range of ambient temperatures in order to titrate the cellular amount of small heat shock proteins according to the cellular demand

Summary

Introduction

Changes in ambient temperature affect the integrity and performance of many cellular structures and processes. Ribonucleicacid thermometers (RNATs) are examples of thermally regulated RNA elements [1] that are located in the 5 -UTR of bacterial messenger RNAs coding for virulence [2,3,4,5,6], cold[7] and heat-shock genes [8,9,10] They operate at the posttranscriptional level and alter the accessibility of the ribosome binding site (RBS) to the ribosomal initiation complex in response to temperature changes: in the off-state, a complimentary sequence located in the 5 -UTR sequesters the RBS in a helix, while in the on-state the RBS locally melts and is released to facilitate translation initiation [11]. Structural diversity in the flanking region of the RBS trapping helix has evolved and topologies of naturally occurring RNATs range from short hairpins to complex multi-helix assemblies with critical tertiary interaction (an overview can be found in [11])

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