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

Abstract

Neisseria meningitidis causes bacterial meningitis and septicemia. It evades the host complement system by upregulating expression of immune evasion factors in response to changes in temperature. RNA thermometers within mRNAs control expression of bacterial immune evasion factors, including CssA, in the 5′-untranslated region of the operon for capsule biosynthesis. We dissect the molecular mechanisms of thermoregulation and report the structure of the CssA thermometer. We show that the RNA thermometer acts as a rheostat, whose stability is optimized to respond in a small temperature range around 37°C as occur within the upper airways during infection. Small increases in temperature gradually open up the structure to allow progressively increased access to the ribosome binding site. Even small changes in stability induced by mutations of imperfect base pairs, as in naturally occurring polymorphisms, shift the thermometer response outside of the desired temperature range, suggesting that its activity could be modulated by pharmacological intervention.

Applications of NMR to structure determination of RNAs large and small

“…In several cases, these were determined using experimental information beyond NMR. For example, for the CssA thermometer [31], J-K region of EMCV IRES RNA [124] and yeast U2/U6 complex [125], small angle X-ray scattering (SAXS) data were used to validate the structure or in the structure calculation itself. In the case of the 131 nts conserved retroviral RNA packaging element, cryo-EM was used along with NMR constraints to determine/validate the NMR structure [43].…”

“…We illustrate this simple but effective strategy using our recent work on a 110 nts RNA thermometer from Neisseria meningitides [31], of which 70 nts comprise the functional and folded core. This approach allowed us to obtain complete assignments and a large number of experimental constraints that would have been impossible if we had studied the complete RNA due to its large asymmetry leading to short T2 relaxation and spectral overlap.…”

“…Unlike proteins, full deuteration is not a viable option for large RNAs, because there are too few NH’s to establish any structural property beyond base pairing. Selective base labeling by residue or resonance type leads to simpler NMR spectra and only requires one of many labeled nucleotide triphosphates (NTPs) prepared by chemical or chemi-enzymatic methods, mixed in with the remaining unlabeled NTPs during in vitro enzymatic transcription [31–35]. For example, multiple nucleotide-specific 13 C, 15 N labeled and specific deuterated nucleotide labeled samples were prepared to investigate the structures of the 56 nts tRNApro; the 68 nts U5 primer binding site (U5-PBS) domain of murine leukemia virus (MLV) RNA; the 63 nts pseudoknot from MLV and the 68 nts core of the RNA thermometer from Neisseria meningitidis [31–33,36].…”

“…Deuteration of the H3′, H4′ and H5′/5′ positions in the sugar and H5 position in C/U can be done straightforwardly using commercially available NTPs [37,38]. This method was successfully applied to determine several interesting RNA structures [31,39]. …”

Interaction and signalling networks: a report from the fourth ‘Young Microbiologists Symposium on Microbe Signalling, Organisation and Pathogenesis’

“…His group discovered and characterized the RNA thermometer CssA from Neisseria meningitidis, an elegant mechanism that this microbe uses to adapt to different temperature changes. Christoph explained how, using NMR spectroscopy and SHAPE (selective 2¢-OH acylation analysed by primer extension) assays, the group discovered that, at low temperature (30 C), all base pair regions of CssA are stably formed, and the ribosome cannot access the RBS which is fully occluded [21]. As the temperature is raised, the RNA structure starts to unfold and, by 42 C, the thermometer structure is fully open, leading to efficient translation.…”