Mn(2+)-Sensing Mechanisms of yybP-ykoY Orphan Riboswitches (original) (raw)
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Local-to-global signal transduction at the core of the Mn2+ sensing riboswitch
The widespread manganese-ion sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch from Xanthomonas oryzae at 2.85 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >50 μs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified...
The Ubiquitous yybP-ykoY Riboswitch Is a Manganese-Responsive Regulatory Element
Molecular Cell, 2015
Highlights d Both transcription and translation of the Mn 2+ exporter MntP are induced by Mn 2+ d The Fur and MntR repressors activate mntP transcription by antagonizing H-NS d Mn 2+ binding to the yybP-ykoY motif in the mntP 5 0 UTR leads to increased translation d Other E. coli and B. subtilis genes with the yybP-ykoY motif also respond to Mn 2+
Biochemistry, 1998
Based on its metallo-cofactor, the manganese-dependent ribonucleotide reductase (Mn-RRase) responsible for delivery of DNA precursors in the Mn-requiring Gram-positive bacterium Corynebacterium (formerly BreVibacterium) ammoniagenes ATCC 6872 is no longer considered as a simple analogue of the aerobic Fe-RRase of Escherichia coli but as the prototype of the class IV enzymes (1). Deliberate dissociation of the Mn-RRase holoenzyme and an improved sample preparation of the dimeric CA2 protein allowed further characterization of the inherent metallo-cofactor by Q-band electron paramagnetic resonance (EPR) spectroscopy. At 40 K, a distinct hyperfine sextet (I ) 5 / 2 , 55 Mn) pattern with a weak zero-field splitting was detected in the CA2 protein prepared from manganese-sufficient cells displaying high RRase activity as expected. This Q-band Mn(II) signal was absent in the apo-CA2 protein obtained from manganese-depleted cells devoid of this enzymatic activity. The presence of a mixed valence manganese cluster in the C. ammoniagenes RRase is excluded since no complex multiline EPR signals were detected in the CA2 protein even at very low (8 K) temperature. The observed Mn(II) spectrum indicates a proteinbound manganese which was modified in the presence of 5.7 mM p-methoxyphenol, but is insensitive toward 10 mM EDTA. Thus, the manganese appeared to be either strictly bound or buried within a hydrophobic pocket of the CA2 protein, inaccessible for EDTA.
Nucleic Acids Research
Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer–ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently r...
Proceedings of the National Academy of Sciences, 2010
Functionally critical metals interact with RNA through complex coordination schemes that are currently difficult to visualize at the atomic level under solution conditions. Here, we report a new approach that combines NMR and XAS to resolve and characterize metal binding in the most highly conserved P4 helix of ribonuclease P (RNase P), the ribonucleoprotein that catalyzes the divalent metal ion-dependent maturation of the 5′ end of precursor tRNA. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals that the Zn 2þ bound to a P4 helix mimic is sixcoordinate, with an average Zn-O/N bond distance of 2.08 Å. The EXAFS data also show intense outer-shell scattering indicating that the zinc ion has inner-shell interactions with one or more RNA ligands. NMR Mn 2þ paramagnetic line broadening experiments reveal strong metal localization at residues corresponding to G378 and G379 in B. subtilis RNase P. A new "metal cocktail" chemical shift perturbation strategy involving titrations with CoðNH 3 Þ 3þ 6 , Zn 2þ , and CoðNH 3 Þ 3þ 6 ∕Zn 2þ confirm an inner-sphere metal interaction with residues G378 and G379. These studies present a unique picture of how metals coordinate to the putative RNase P active site in solution, and shed light on the environment of an essential metal ion in RNase P. Our experimental approach presents a general method for identifying and characterizing inner-sphere metal ion binding sites in RNA in solution.
Metal Binding Studies and EPR Spectroscopy of the Manganese Transport Regulator MntR †
Biochemistry, 2006
Manganese transport regulator (MntR) is a member of the diphtheria toxin repressor (DtxR) family of transcription factors that is responsible for manganese homeostasis in Bacillus subtilis. Prior biophysical studies have focused on the metal-mediated DNA binding of MntR [Lieser, S. A., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2003) Biochemistry 42, 12634-12642], as well as metal stabilization of the MntR structure [Golynskiy, M. V., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2005) Biochemistry 44, 3380-3389], but only limited data on the metal-binding affinities for MntR are available.
Journal of Molecular Biology, 2010
The ulaG gene, located in the ula regulon, is crucial for the catabolism of L-ascorbate under anaerobic conditions and it has been proposed to encode for the putative L-ascorbate-6-P lactonase. The ulaG gene is widespread among eubacteria, including human commensal and pathogenic genera such as Escherichia, Shigella, Klebsiella and Salmonella. Here, we report the three-dimensional structures of the apoenzyme and Mn 2+ holoenzyme of UlaG from E. coli to 2.6 Å resolution, determined using single-wavelength anomalous diffraction phasing and molecular replacement, respectively. The structures reveal a highly specialized metallo-β-lactamase-like fold derived from an ancient structural template that was involved in RNA maturation and DNA repair. This fold has a novel quaternary architecture consisting of a hexameric ring formed by a trimer of UlaG dimers. A mononuclear Mn 2+ -binding site resides at the core of the active site, which displays micromolar affinity for Mn 2+ and a distorted trigonal bipyramidal coordination. The active site Mn 2+ ion can be replaced by Co 2+ or Zn 2+ , but not by Fe 3+ . We further show that the Mn 2+ or Co 2+ -loaded enzyme exhibits lactonase activity towards L-ascorbate 6-P, thereby providing the first direct evidence of its catalytic role in the L-ascorbate catabolic pathway. Guided by the structural homology, we show that UlaG is able to cleave phosphodiester linkages in cyclic nucleotides, suggesting that the conservation of the fold and of the key catalytic residues allows for the evolutionary acquisition of substrate specificity for novel but related substrates.
Roles of the A and C Sites in the Manganese-Specific Activation of MntR
Biochemistry, 2013
The manganese transport regulator (MntR) represses the expression of genes involved in manganese uptake in Bacillus subtilis. It selectively responds to Mn 2+ and Cd 2+ over other divalent metal cations including Fe 2+ , Co 2+ and Zn 2+. Previous work has shown that MntR forms binuclear complexes with Mn 2+ or Cd 2+ at two binding sites, labeled A and C, that are separated by 4.4 Å. Zinc activates MntR poorly and binds only to the A site, forming a mononuclear complex. The difference in metal binding stoichiometry suggested a mechanism for selectivity in MntR. Larger metal cations are strongly activating because they can form the binuclear complex, while smaller metal ions cannot bind with the geometry needed to fully occupy both metal-binding sites. To investigate this hypothesis, structures of MntR in complex with two other non-cognate metal ions, Fe 2+ and Co 2+ , have been solved. Each metal forms a mononuclear complex with MntR with the metal ion bound in the A site, supporting the conclusions drawn from the Zn 2+ complex. Additionally, we investigated two site-specific mutants of MntR, E11K and H77A, that contain substitutions to metal binding residues in the A site. While metal binding in each mutant is significantly altered relative to wild-type MntR, both mutants retain activity and selectivity for Mn 2+ in vitro and in vivo. That observation, coupled with previous studies, suggests that the A and C sites both contribute to the selectivity of MntR.
Appl Magn Reson, 2007
The catalytic activity of the tertiary stabilized hammerhead ribozyme (tsHHR.z) is by three orders of magnitude higher than the one of the long-known minimal construct (mHHRz). This gives rise to the question whether the single high-affŸ manganese(lI) binding site present in both ribozymes is located closer to the cleavage site and the transition state in the tsHHRz than in the mHHRz, which would make a direct involvement of this metal(II) ion in the bond-breaking step more likely. Here, we used W-band 3~P-Davies-ENDOR (electron-nuclear double resonance) to complement earlier reported L4N-ESEEM/HYSCORE (electron spin echo envelope modulation/hyperfine sublevel correlation) studies. The 3tP-ENDOR spectrum of the mHHRz revealed a doublet with a splitting of 8.4(+_0.5) MHz but unresolved hyperfine anisotropy. Such a large splitting indicates ah inner-sphere coordination of a phosphate backbone group with a significant amount of spin density on the phosphorous nucleus. This is in good agreement with the 3~p isotropic hyperfine constant, A~~o(3~P), of +7.8 MHz obtained by density functional theory calculations on the structure of the Mn 2 § binding site as found in crystals of the same ribozyme. This supports the idea that the structure and location of the binding site in the mHHRz is in frozen buffer similar to that found in the crystal. Since the W-band ENDOR spectrum of the tsHHRz also shows a 3~p splitting of 8.4(+_0.5) MHz, the local structures of both binding sites appear to be similar, which agrees with the coincidence of the)4N data. Ah involvement of the high-affinity Mn 2 § ion in the catalytic step seems therefore unlikely.