SHAPE analysis of the htrA RNA thermometer from Salmonella enterica

Abstract

RNA thermometers regulate expression of some genes involved in virulence of pathogenic bacteria such as ,, and They often function through temperature-dependent conformational changes that alter accessibility of the ribosome-binding site. The 5'-untranslated region (UTR) of the mRNA from contains a very short RNA thermometer. We have systematically characterized the structure and dynamics of this thermometer at single-nucleotide resolution using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) assays. Our results confirm that the thermometer adopts the predicted hairpin conformation at low temperatures, with conformational change occurring over a physiological temperature regime. Detailed SHAPE melting curves for individual nucleotides suggest that the thermometer unfolds in a cooperative fashion, with nucleotides from both upper and lower portions of the stem gaining flexibility at a common transition temperature. Intriguingly, analysis of an extended 5' UTR sequence revealed not only the presence of the RNA thermometer, but also an additional, stable upstream structure. We generated and analyzed point mutants of the thermometer, revealing elements that modulate its stability, allowing the hairpin to melt under the slightly elevated temperatures experienced during the infection of a warm-blooded host. This work sheds light on structure-function relationships in and related thermometers, and it also illustrates the utility of SHAPE assays for detailed study of RNA thermometer systems.

Characterizing the Structure–Function Relationship of a Naturally Occurring RNA Thermometer

“…Specifically, these experiments represent the first in vivo RNA structure probing of an RNA thermometer and nicely complement previous in vitro structural studies of fourU thermometers. 13,19–21…”

“…Here we sought to better understand the mechanistic details of the agsA fourU thermometer by studying for the first time how in vivo structural changes couple to gene expression. In recent work, SHAPE-based probing has been deployed to explore similar RNA thermometer mechanisms in vitro 21,22 . Now, by combining in-cell SHAPE-Seq to probe RNA structures in vivo , fluorescent reporter measurements of gene expression, and experimentally restrained computational structure prediction, we present a detailed view of the agsA thermometer structural ensemble and its response to heat shock.…”

High-throughput determination of RNA structures

“…The implication is that if the rate constants of the chemical reaction are known, then reactivities can be interpreted as quantitative kinetic parameters about nucleotide-level RNA structural properties. For example, when defined in this way, reactivity values that are in between the extreme high and low ends of the scale reveal information about nuanced structural contexts such as base stacking 70 , slow structural fluctuations in specific regions of the molecules 25,74 , regions of the molecule which undergo melting transitions 69,96,97 , tertiary interactions 69,71,72,98 , and others, which highlight the richness of chemical probing reactivity data.…”

RNA Secondary Structurome Revealed Distinct Thermoregulation in Plasmodium falciparum

“…Recent studies ( Mustoe et al, 2018 ) have established that the RNA secondary and tertiary structures act as fundamental and pervasive regulators of individual transcript expression within seconds ( Ganser et al, 2019 ). The control of gene expression in organisms through structural transformation of RNA elements can promote rapid responses to changing environmental conditions and enhance regulation at the transcription level ( Choi et al, 2017 ). For example RNA thermometers (RNATs), which are usually located in the 5′-UTR region, can respond to changes in temperature within seconds and directly control nascent or existing mRNA translation.…”