Martin Hengesbach

Single-Molecule FRET Reveals the Folding Dynamics of the Human Telomerase RNA Pseudoknot Domain†

Telomerase catalyzed synthesis of telomere DNA provides the foundation for DNA-protein structures called telomeres. Telomeres are required to evade DNA processing which may result in nucleolytic degradation and fusion of linear chromosomes. Human telomerase contains a protein reverse transcriptase (hTERT), telomerase RNA (hTR), and several additional proteins.[1] hTR provides the template sequence for hTERT during a novel reverse transcription reaction that produces short telomere DNA repeat sequences.[2] Additionally, hTR provides a flexible scaffold for the assembly and function of telomerase associated proteins[3] and has recently been suggested to contribute to enzyme catalysis.[4] Many pathogenic mutations have been mapped to hTR;[5] however the precise mechanisms of these mutations remain unclear.

Rapid NMR screening of RNA secondary structure and binding

Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3′ end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.

Single-Molecule FRET Reveals a Cooperative Effect of Two Methyl Group Modifications in the Folding of Human Mitochondrial tRNALys

Using a combination of advanced RNA synthesis techniques and single molecule spectroscopy, the deconvolution of individual contributions of posttranscriptional modifications to the overall folding and stabilization of human mitochondrial tRNA(Lys) is described. An unexpected destabilizing effect of two pseudouridines on the native tRNA folding was evidenced. Furthermore, the presence of m(2)G10 alone does not facilitate the folding of tRNA(Lys), but a stabilization of the biologically functional cloverleaf shape in conjunction with the principal stabilizing component m(1)A9 exceeds the contribution of m(1)A alone. This constitutes an unprecedented cooperative effect of two nucleotide modifications in the context of a naturally occurring RNA, which may be of general importance for tRNA structure and help understanding several recently described decay pathways for hypomodified tRNAs.

RNA Intramolecular Dynamics by Single‐Molecule FRET

Investigation of single RNA molecules using fluorescence resonance energy transfer (FRET) is a powerful approach to investigate dynamic and thermodynamic aspects of the folding process of a given RNA. Its application requires interdisciplinary work from the fields of chemistry, biochemistry, and physics. The present work gives detailed instructions on the synthesis of RNA molecules labeled with two fluorescent dyes interacting by FRET, as well as on their investigation by single‐molecule fluorescence spectroscopy. Curr. Protoc. Nucleic Acid Chem. 34:11.12.1‐11.12.22.

Single-molecule analysis of telomerase structure and function

The telomerase ribonucleoprotein is a specialized reverse transcriptase required to maintain protective chromosome end-capping structures called telomeres. In most cells, telomerase is not active and the natural shortening of telomeres with each round of DNA replication ultimately triggers cell growth arrest. In contrast, the presence of telomerase confers a high level of renewal capacity upon rapidly dividing cells. Telomerase is aberrantly activated in 90% of human cancers and thus represents an important target for anticancer therapeutics. However, the naturally low abundance of telomerase has hampered efforts to obtain high-resolution models for telomerase structure and function. To circumvent these challenges, single-molecule techniques have recently been employed to investigate telomerase assembly, structure, and catalysis.

Single-molecule FRET studies of counterion effects on the free energy landscape of human mitochondrial lysine tRNA.

The folding energy landscape of RNA is greatly affected by interactions between the RNA and counterions that neutralize the backbone negative charges and may also participate in tertiary contacts. Valence, size, coordination number, and electron shell structure can all contribute to the energetic stabilization of specific RNA conformations. Using single-molecule fluorescence resonance energy transfer (smFRET), we have examined the folding properties of the RNA transcript of human mitochondrial tRNA(Lys), which possesses two different folded states in addition to the unfolded one under conditions of thermodynamic equilibrium. We have quantitatively analyzed the degree of RNA tertiary structure stabilization for different types of cations based on a thermodynamic model that accounts for multiple conformational states and RNA-ion interactions within each state. We have observed that small monovalent ions stabilize the tRNA tertiary structure more efficiently than larger ones. More ions were found in close vicinity of compact RNA structures, independent of the type of ion. The largest conformation-dependent binding specificity of ions of the same charge was found for divalent ions, for which the ionic radii and coordination properties were responsible for shaping the folding free energy.

Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET

Pseudouridine is the most abundant of more than 100 chemically distinct natural ribonucleotide modifications. Its synthesis consists of an isomerization reaction of a uridine residue in the RNA chain and is catalyzed by pseudouridine synthases. The unusual reaction mechanism has become the object of renewed research effort, frequently involving replacement of the substrate uridines with 5-fluorouracil (f(5)U). f(5)U is known to be a potent inhibitor of pseudouridine synthase activity, but the effect varies among the target pseudouridine synthases. Derivatives of f(5)U have previously been detected, which are thought to be either hydrolysis products of covalent enzyme-RNA adducts, or isomerization intermediates. Here we describe the interaction of pseudouridine synthase 1 (Pus1p) with f(5)U-containing tRNA. The interaction described is specific to Pus1p and position 27 in the tRNA anticodon stem, but the enzyme neither forms a covalent adduct nor stalls at a previously identified reaction intermediate of f(5)U. The f(5)U27 residue, as analyzed by a DNAzyme-based assay using TLC and mass spectrometry, displayed physicochemical properties unaltered by the reversible interaction with Pus1p. Thus, Pus1p binds an f(5)U-containing substrate, but, in contrast to other pseudouridine synthases, leaves the chemical structure of f(5)U unchanged. The specific, but nonproductive, interaction demonstrated here thus constitutes an intermediate of Pus turnover, stalled by the presence of f(5)U in an early state of catalysis. Observation of the interaction of Pus1p with fluorescence-labeled tRNA by a real-time readout of fluorescence anisotropy and FRET revealed significant structural distortion of f(5)U-tRNA structure in the stalled intermediate state of pseudouridine catalysis.

Differential Scanning Fluorimetry for Monitoring RNA Stability

Cellular RNA function is closely linked to RNA structure. It is therefore imperative to develop methods that report on structural stability of RNA and how it is modulated by binding of ions, other osmolytes, and RNA‐binding ligands. Here, we present a novel method to analyze the stability of virtually any structured RNA in a highly parallel fashion. This method can easily determine the influence of various additives on RNA stability, and even characterize ligand‐induced stabilization of riboswitch RNA. Current approaches to assess RNA stability include thermal melting profiles (absorption or circular dichroism) and differential scanning calorimetry. These techniques, however, require a substantial amount of material and cannot be significantly parallelized. Current fluorescence spectroscopic methods rely on intercalating dyes, which alter the stability of RNA. We employ the commercial fluorescent dye RiboGreen, which discriminates between single‐stranded (or unstructured regions) and double‐stranded RNA. Binding leads to an increase in fluorescence quantum yield, and thus reports structural changes by a change in fluorescence intensity.

Ligand-modulated folding of the full-length adenine riboswitch probed by NMR and single-molecule FRET spectroscopy

Abstract The full-length translation-regulating add adenine riboswitch (Asw) from Vibrio vulnificus has a more complex conformational space than its isolated aptamer domain. In addition to the predicted apo (apoA) and holo conformation that feature the conserved three-way junctional purine riboswitch aptamer, it adopts a second apo (apoB) conformation with a fundamentally different secondary structure. Here, we characterized the ligand-dependent conformational dynamics of the full-length add Asw by NMR and by single-molecule FRET (smFRET) spectroscopy. Both methods revealed an adenine-induced secondary structure switch from the apoB-form to the apoA-form that involves no tertiary structural interactions between aptamer and expression platform. This strongly suggests that the add Asw triggers translation by capturing the apoA-form secondary structure in the holo state. Intriguingly, NMR indicated a homogenous, docked aptamer kissing loop fold for apoA and holo, while smFRET showed persistent aptamer kissing loop docking dynamics between comparably stable, undocked and docked substates of the apoA and the holo conformation. Unraveling the folding of large junctional riboswitches thus requires the integration of complementary solution structural techniques such as NMR and smFRET.

EXPLORING THE FOLDING FREE ENERGY LANDSCAPE OF SMALL RNA MOLECULES BY SINGLE-PAIR FÖRSTER RESONANCE ENERGY TRANSFER

Proteins and RNA are biological macromolecules built from linear polymers. The process by which they fold into compact, well-defined, three-dimensional architectures to perform their functional tasks is still not well understood. It can be visualized by Brownian motion of an ensemble of molecules through a rugged energy landscape in search of an energy minimum corresponding to the native state. To explore the conformational energy landscape of small RNAs, single pair Forster resonance energy transfer (spFRET) experiments on solutions as well as on surface-immobilized samples have provided new insights. In this review, we focus on our recent work on two FRET-labeled small RNAs, the Diels-Alderase (DAse) ribozyme and the human mitochondrial tRNALys. For both RNAs, three different conformational states can be distinguished, and the associated mean FRET efficiencies provide clues about their structural properties. The systematic variation of their free energies with the concentration of Mg2+ counterions was a...