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Influence of ground-state structure and Mg2+ binding on folding kinetics of the guanine-sensing riboswitch aptamer domain
(2011)
- Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop–loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gswloop) in the absence of Mg2+. However, if Mg2+ is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop–loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gswloop is tunable through variation of the Mg2+ concentration. We quantitatively describe the influence of distinct Mg2+ concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution.
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Modulation of the stability of the Salmonella fourU-type RNA thermometer
(2011)
- RNA thermometers are translational control elements that regulate the expression of bacterial heat shock and virulence genes. They fold into complex secondary structures that block translation at low temperatures. A temperature increase releases the ribosome binding site and thus permits translation initiation. In fourU-type RNA thermometers, the AGGA sequence of the SD region is paired with four consecutive uridines. We investigated the melting points of the wild-type and mutant sequences. It was decreased by 5°C when a stabilizing GC basepair was exchanged by an AU pair or increased by 11°C when an internal AG mismatch was converted to a GC pair, respectively. Stabilized or destabilized RNA structures are directly correlated with decreased or increased in vivo gene expression, respectively. Mg2+ also affected the melting point of the fourU thermometer. Variations of the Mg2+ concentration in the physiological range between 1 and 2 mM translated into a 2.8°C shift of the melting point. Thus, Mg2+ binding to the hairpin RNA is regulatory relevant. Applying three different NMR techniques, two Mg2+ binding sites were found in the hairpin structure. One of these binding sites could be identified as outer sphere binding site that is located within the fourU motif. Binding of the two Mg2+ ions exhibits a positive cooperativity with a Hill coefficient of 1.47. Free energy values delta G for Mg2+ binding determined by NMR are in agreement with data determined from CD measurements. Physiological Mg2+ concentrations reduce enthalpy and entropy values of uncorrelated base pair opening processes for almost all nucleobases.
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Structure and dynamics of the deoxyguanosine-sensing riboswitch studied by NMR-spectroscopy
(2011)
- The mfl-riboswitch regulates expression of ribonucleotide reductase subunit in Mesoplasma florum by binding to 2´-deoxyguanosine and thereby promoting transcription termination. We characterized the structure of the ligand-bound aptamer domain by NMR spectroscopy and compared the mfl-aptamer to the aptamer domain of the closely related purine-sensing riboswitches. We show that the mfl-aptamer accommodates the extra 2´-deoxyribose unit of the ligand by forming a more relaxed binding pocket than these found in the purine-sensing riboswitches. Tertiary structures of the xpt-aptamer bound to guanine and of the mfl-aptamer bound to 2´-deoxyguanosine exhibit very similar features, although the sequence of the mfl-aptamer contains several alterations compared to the purine-aptamer consensus sequence. These alterations include the truncation of a hairpin loop which is crucial for complex formation in all purine-sensing riboswitches characterized to date. We further defined structural features and ligand binding requirements of the free mfl-aptamer and found that the presence of Mg2+ is not essential for complex formation, but facilitates ligand binding by promoting pre-organization of key structural motifs in the free aptamer.
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Translation on demand by a simple RNA-based thermosensor
(2011)
- Structured RNA regions are important gene control elements in prokaryotes and eukaryotes. Here, we show that the mRNA of a cyanobacterial heat shock gene contains a built-in thermosensor critical for photosynthetic activity under stress conditions. The exceptionally short 5´-untranslated region is comprised of a single hairpin with an internal asymmetric loop. It inhibits translation of the Synechocystis hsp17 transcript at normal growth conditions, permits translation initiation under stress conditions and shuts down Hsp17 production in the recovery phase. Point mutations that stabilized or destabilized the RNA structure deregulated reporter gene expression in vivo and ribosome binding in vitro. Introduction of such point mutations into the Synechocystis genome produced severe phenotypic defects. Reversible formation of the open and closed structure was beneficial for viability, integrity of the photosystem and oxygen evolution. Continuous production of Hsp17 was detrimental when the stress declined indicating that shutting-off heat shock protein production is an important, previously unrecognized function of RNA thermometers. We discovered a simple biosensor that strictly adjusts the cellular level of a molecular chaperone to the physiological need.
