Exploring the energy landscape of a SAM-I riboswitch

SAM-I riboswitches regulate gene expression through transcription termination upon binding a S-adenosyl-L-methionine (SAM) ligand. In previous work, we characterized the conformational energy landscape of the full-length Bacillus subtilis yitJ SAM-I riboswitch as a function of Mg$^{2+}$ and SAM ligand concentrations. Here, we have extended this work with measurements on a structurally similar ligand, S-adenosyl-L-homocysteine (SAH), which has, however, a much lower binding affinity. Using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling (HMM) analysis, we identified major conformations and determined their fractional populations and dynamics. At high Mg$^{2+}$ concentration, FRET analysis yielded four distinct conformations, which we assigned to two terminator and two antiterminator states. In the same solvent, but with SAM added at saturating concentrations, four states persisted, although their populations, lifetimes and interconversion dynamics changed. In the presence of SAH instead of SAM, HMM revealed again four well-populated states and, in addition, a weakly populated ‘hub’ state that appears to mediate conformational transitions between three of the other states. Our data show pronounced and specific effects of the SAM and SAH ligands on the RNA conformational energy landscape. Interestingly, both SAM and SAH shifted the fractional populations toward terminator folds, but only gradually, so the effect cannot explain the switching action. Instead, we propose that the noticeably accelerated dynamics of interconversion between terminator and antiterminator states upon SAM binding may be essential for control of transcription

A simple route to highly active single-enzyme nanogels

Just add sugar: the synthesis of single-enzyme nanogels, a class of highly robust nanobiocatalysts, is boosted by the addition of carbohydrates. Our methodology is demonstrated with a dozen commercial proteins, spanning a large size interval and a broad domain of applications. In addition, new in-depth structural characterizations are provided.</p

Mg 2þ-dependent folding of a Diels-Alderase ribozyme probed by single-molecule

FRET analysi

Energy Landscape Analysis of the Full-Length SAM-I Riboswitch using Single-Molecule FRET Spectroscopy

Binding of the ligand S-adenosyl-L-methionine (SAM) produces major structural changes in the SAM-I riboswitch (RS) and thereby regulates gene expression via transcription termination. As yet, the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I RS have not been deeply investigated. We have studied its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy. smFRET histograms of differently FRET-labeled constructs were so complicated that they could only be resolved with the help of kinetic experiments on immobilized riboswitches and hidden Markov modeling (HMM) analysis. At least four conformational states were identified, both in the presence and the absence of SAM. We determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds were found to coexist under all conditions; SAM binding induced only a gradual increase in the population of terminator states. Conformational transitions were much faster with bound SAM, which may be crucial for off-switching during the brief decision window before expression of the downstream gene

A methyl group controls conformational equilibrium in human mitochondrial tRNA(Lys)

The unmodified maturation intermediate in the biogenesis of human mitochondrial tRNA(Lys) adopts a nonfunctional conformation, resembling an extended hairpin. Single molecule fluorescence resonance energy transfer (FRET) studies reveal the additional presence of a small population in a functional cloverleaf conformation. Both conformations are in a dynamic equilibrium, interconverting on the 100 ms time scale. The post-transcriptional methylation of adenosine 9 to 1-methyladenosine is a crucial step in the biogenesis of the mature tRNA. This single methyl group, by disfavoring the nonfunctional conformation, shifts the observed equilibrium toward the functional cloverleaf and makes it available for further maturation. Here, single molecule FRET analysis makes dynamics and thermodynamics of these small RNAs conveniently accessible and elucidates the mode of action of a simple post-transcriptional modification.status: publishe

Time-resolved Photoluminescence and Theoretical Study of Excitons in PTCDA

doctor rerum naturalium (Dr. rer. nat.) vorgelegt von M. S. Andrei Yu. Kobitski geboren am 18.10.76 in Schwerin eingereicht am...................... Gutachter

Mg concentration dependence of the average inter-dye distance (filled squares) and the width of the distance distribution (open squares) of the state

<p><b>Copyright information:</b></p><p>Taken from "Mg-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis"</p><p></p><p>Nucleic Acids Research 2007;35(6):2047-2059.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874616.</p><p>© 2007 The Author(s)</p> Solid lines represent a global fit according to the Hill equation, yielding a midpoint Mg concentration of (3.8 ± 0.5) mM and a cooperativity parameter = 4.2 ± 0.8

Histograms of FRET efficiency values, , taken from single molecules exposed to buffer solutions with 0, 0

<p><b>Copyright information:</b></p><p>Taken from "Mg-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis"</p><p></p><p>Nucleic Acids Research 2007;35(6):2047-2059.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874616.</p><p>© 2007 The Author(s)</p>625, 1.25, 2.5, 5, 10, 20, 40 and 100 mM Mg concentration. The lowest panel shows the histogram for molecules exposed to 0 mM Mg immediately after exposure to 40 mM Mg. Three subpopulations, denoted as (〈〉 ∼ 0), (〈〉 ∼ 0.7) and (〈〉 ∼ 0.9), can be distinguished. Dotted, dashed and solid thick lines represent fit results using model distributions for the and states, respectively. The solid line represents the sum over the three distributions

Thermodynamic scheme describing the Mg-dependent folding of DAse ribozyme

<p><b>Copyright information:</b></p><p>Taken from "Mg-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis"</p><p></p><p>Nucleic Acids Research 2007;35(6):2047-2059.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874616.</p><p>© 2007 The Author(s)</p> () Scheme involving three Mg-free states, denoted as , and , and three Mg-bound states, denoted as , and . Folding is induced by Mg-dependent equilibrium coefficients and () Free energy diagram of the observed populations of ( + ), ( + ) and ( + ) is shown for 0 and 40 mM Mg