Monitoring RNA dynamics in native transcriptional complexes.

Cotranscriptional RNA folding is crucial for the timely control of biological processes, but because of its transient nature, its study has remained challenging. While single-molecule Förster resonance energy transfer (smFRET) is unique to investigate transient RNA structures, its application to cotranscriptional studies has been limited to nonnative systems lacking RNA polymerase (RNAP)-dependent features, which are crucial for gene regulation. Here, we present an approach that enables site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes. By monitoring Escherichia coli nascent riboswitches, we reveal an inverse relationship between elongation speed and metabolite-sensing efficiency and show that pause sites upstream of the translation start codon delimit a sequence hotspot for metabolite sensing during transcription. Furthermore, we demonstrate a crucial role of the bacterial RNAP actively delaying the formation, within the hotspot sequence, of competing structures precluding metabolite binding. Our approach allows the investigation of cotranscriptional regulatory mechanisms in bacterial and eukaryotic elongation complexes.

Fatty acids may influence insulin dynamics through modulation of albumin-Zn2+ interactions.

Insulin is stored within the pancreas in an inactive Zn2+ -bound hexameric form prior to release. Similarly, clinical insulins contain Zn2+ and form multimeric complexes. Upon release from the pancreas or upon injection, insulin only becomes active once Zn2+ disengages from the complex. In plasma and other extracellular fluids, the majority of Zn2+ is bound to human serum albumin (HSA), which plays a vital role in controlling insulin pharmacodynamics by enabling removal of Zn2+ . The Zn2+ -binding properties of HSA are attenuated by non-esterified fatty acids (NEFAs) also transported by HSA. Elevated NEFA concentrations are associated with obesity and type 2 diabetes. Here we present the hypothesis that higher NEFA levels in obese and/or diabetic individuals may contribute to insulin resistance and affect therapeutic insulin dose-response profiles, through modulation of HSA/Zn2+ dynamics. We envisage this novel concept to have important implications for personalized treatments and management of diabetes-related conditions in the future.

A structural intermediate pre-organizes the add adenine riboswitch for ligand recognition.

Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.

Asymmetric base-pair opening drives helicase unwinding dynamics.

The opening of a Watson-Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix.

Unprecedented tunability of riboswitch structure and regulatory function by sub-millimolar variations in physiological Mg2.

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 respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.

Mechanism of DNA loading by the DNA repair helicase XPD.

The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5' to 3' helicase with an essential iron-sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD.

High-affinity RNA binding by a hyperthermophilic single-stranded DNA-binding protein.

Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event.

Binding dynamics of a monomeric SSB protein to DNA: a single-molecule multi-process approach.

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all organisms and are characterized by the presence of an OB (oligonucleotide/oligosaccharide/oligopeptide) binding motif to recognize single-stranded DNA (ssDNA). Despite their critical role in genome maintenance, our knowledge about SSB function is limited to proteins containing multiple OB-domains and little is known about single OB-folds interacting with ssDNA. Sulfolobus solfataricus SSB (SsoSSB) contains a single OB-fold and being the simplest representative of the SSB-family may serve as a model to understand fundamental aspects of SSB:DNA interactions. Here, we introduce a novel approach based on the competition between Förster resonance energy transfer (FRET), protein-induced fluorescence enhancement (PIFE) and quenching to dissect SsoSSB binding dynamics at single-monomer resolution. We demonstrate that SsoSSB follows a monomer-by-monomer binding mechanism that involves a positive-cooperativity component between adjacent monomers. We found that SsoSSB dynamic behaviour is closer to that of Replication Protein A than to Escherichia coli SSB; a feature that might be inherited from the structural analogies of their DNA-binding domains. We hypothesize that SsoSSB has developed a balance between high-density binding and a highly dynamic interaction with ssDNA to ensure efficient protection of the genome but still allow access to ssDNA during vital cellular processes.

Morphology-Specific Inhibition of ß-Amyloid Aggregates by 17ß-Hydroxysteroid Dehydrogenase Type 10.

A major hallmark of Alzheimer's disease (AD) is the formation of toxic aggregates of the ß-amyloid peptide (Aß). Given that Aß peptides are known to localise within mitochondria and interact with 17ß-HSD10, a mitochondrial protein expressed at high levels in AD brains, we investigated the inhibitory potential of 17ß-HSD10 against Aß aggregation under a range of physiological conditions. Fluorescence self-quenching (FSQ) of Aß(1-42) labelled with HiLyte Fluor 555 was used to evaluate the inhibitory effect under conditions established to grow distinct Aß morphologies. 17ß-HSD10 preferentially inhibits the formation of globular and fibrillar-like structures but has no effect on the growth of amorphous plaque-like aggregates at endosomal pH 6. This work provides insights into the dependence of the Aß-17ß-HSD10 interaction with the morphology of Aß aggregates and how this impacts enzymatic function.

Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates.

Flap endonuclease 1 (Fen1) is a highly conserved structure-specific nuclease that catalyses a specific incision to remove 5' flaps in double-stranded DNA substrates. Fen1 plays an essential role in key cellular processes, such as DNA replication and repair, and mutations that compromise Fen1 expression levels or activity have severe health implications in humans. The nuclease activity of Fen1 and other FEN family members can be stimulated by processivity clamps such as proliferating cell nuclear antigen (PCNA); however, the exact mechanism of PCNA activation is currently unknown. Here, we have used a combination of ensemble and single-molecule Förster resonance energy transfer together with protein-induced fluorescence enhancement to uncouple and investigate the substrate recognition and catalytic steps of Fen1 and Fen1/PCNA complexes. We propose a model in which upon Fen1 binding, a highly dynamic substrate is bent and locked into an open flap conformation where specific Fen1/DNA interactions can be established. PCNA enhances Fen1 recognition of the DNA substrate by further promoting the open flap conformation in a step that may involve facilitated threading of the 5' ssDNA flap. Merging our data with existing crystallographic and molecular dynamics simulations we provide a solution-based model for the Fen1/PCNA/DNA ternary complex.

Towards ratiometric sensing of amyloid fibrils in vitro.

The aggregation of amyloid-ß peptide and its accumulation in the human brain has an important role in the etiology of Alzheimer's disease. Thioflavin T has been widely used as a fluorescent marker for these amyloid aggregates. Nevertheless, its complex photophysical behavior, with strong wavelength dependencies of all its fluorescence properties, requires searching for new fluorescent probes. The use of 2-(2'-hydroxyphenyl)imidazo[4,5-b]pyridine (HPIP), which shows two emission bands and a rich excited-state behavior due to the existence of excited-state intramolecular processes of proton transfer and charge transfer, is proposed. These properties result in a high sensitivity of HPIP fluorescence to its microenvironment and cause a large differential fluorescence enhancement of the two bands upon binding to aggregates of the amyloid-ß peptide. Based on this behavior, a very sensitive ratiometric method is established for the detection and quantification of amyloid fibrils, which can be combined with the monitoring of fluorescence anisotropy. The binding selectivity of HPIP is discussed on the basis of the apparent binding equilibrium constants of this probe to amyloid-ß (1-42) fibrils and to the nonfibrillar protein bovine serum albumin. Finally, an exhaustive comparison between HPIP and thioflavin T is presented to discuss the sensitivity and specificity of these probes to amyloid aggregates and the significant advantages of the HPIP dye for quantitative determinations.

Single-molecule chemical denaturation of riboswitches.

To date, single-molecule RNA science has been developed almost exclusively around the effect of metal ions as folding promoters and stabilizers of the RNA structure. Here, we introduce a novel strategy that combines single-molecule Förster resonance energy transfer (FRET) and chemical denaturation to observe and manipulate RNA dynamics. We demonstrate that the competing interplay between metal ions and denaturant agents provides a platform to extract information that otherwise will remain hidden with current methods. Using the adenine-sensing riboswitch aptamer as a model, we provide strong evidence for a rate-limiting folding step of the aptamer domain being modulated through ligand binding, a feature that is important for regulation of the controlled gene. In the absence of ligand, the rate-determining step is dominated by the formation of long-range key tertiary contacts between peripheral stem-loop elements. In contrast, when the adenine ligand interacts with partially folded messenger RNAs, the aptamer requires specifically bound Mg(2+) ions, as those observed in the crystal structure, to progress further towards the native form. Moreover, despite that the ligand-free and ligand-bound states are indistinguishable by FRET, their different stability against urea-induced denaturation allowed us to discriminate them, even when they coexist within a single FRET trajectory; a feature not accessible by existing methods.

Cotranscriptional Folding of a 5' Stem-loop in the Escherichia coli tbpA Riboswitch at Single-nucleotide Resolution.

Transcription elongation is one of the most important processes in the cell. During RNA polymerase elongation, the folding of nascent transcripts plays crucial roles in the genetic decision. Bacterial riboswitches are prime examples of RNA regulators that control gene expression by altering their structure upon metabolite sensing. It was previously revealed that the thiamin pyrophosphate-sensing tbpA riboswitch in Escherichia coli cotranscriptionally adopts three main structures leading to metabolite sensing. Here, using single-molecule FRET, we characterize the transition in which the first nascent structure, a 5' stem-loop, is unfolded during transcription elongation to form the ligand-binding competent structure. Our results suggest that the structural transition occurs in a relatively abrupt manner, i.e., within a 1-2 nucleotide window. Furthermore, a highly dynamic structural exchange is observed, indicating that riboswitch transcripts perform rapid sampling of nascent co-occurring structures. We also observe that the presence of the RNAP stabilizes the 5' stem-loop along the elongation process, consistent with RNAP interacting with the 5' stem-loop. Our study emphasizes the role of early folding stem-loop structures in the cotranscriptional formation of complex RNA molecules involved in genetic regulation.

Solution-based single molecule imaging of surface-immobilized conjugated polymers.

The photophysical behavior of conjugated polymers used in modern optoelectronic devices is strongly influenced by their structural dynamics and conformational heterogeneity, both of which are dependent on solvent properties. Single molecule studies of these polymer systems embedded in a host matrix have proven to be very powerful to investigate the fundamental fluorescent properties. However, such studies lack the possibility of examining the relationship between conformational dynamics and photophysical response in solution, which is the phase from which films for devices are deposited. By developing a synthetic strategy to incorporate a biotin moiety as a surface attachment point at one end of a polyalkylthiophene, we immobilize it, enabling us to make the first single molecule fluorescence measurements of conjugated polymers for long periods of time in solution. We identify fluctuation patterns in the fluorescence signal that can be rationalized in terms of photobleaching and stochastic transitions to reversible dark states. Moreover, by using the advantages of solution-based imaging, we demonstrate that the addition of oxygen scavengers improves optical stability by significantly decreasing the photobleaching rates.

Molecular insights into the ligand-controlled organization of the SAM-I riboswitch.

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.

Constitutive regulatory activity of an evolutionarily excluded riboswitch variant.

The exquisite specificity of the adenine-responsive riboswitch toward its cognate metabolite has been shown to arise from the formation of a Watson-Crick interaction between the adenine ligand and residue U65. A recent crystal structure of a U65C adenine aptamer variant has provided a rationale for the phylogenetic conservation observed at position 39 for purine aptamers. The G39-C65 variant adopts a compact ligand-free structure in which G39 is accommodated by the ligand binding site and is base-paired to the cytosine at position 65. Here, we demonstrate using a combination of biochemical and biophysical techniques that the G39-C65 base pair not only severely impairs ligand binding but also disrupts the functioning of the riboswitch in vivo by constitutively activating gene expression. Folding studies using single-molecule FRET revealed that the G39-C65 variant displays a low level of dynamic heterogeneity, a feature reminiscent of ligand-bound wild-type complexes. A restricted conformational freedom together with an ability to significantly fold in monovalent ions are exclusive to the G39-C65 variant. This work provides a mechanistic framework to rationalize the evolutionary exclusion of certain nucleotide combinations in favor of sequences that preserve ligand binding and gene regulation functionalities.

Folding of the SAM-I riboswitch: a tale with a twist.

Riboswitches are ligand-dependent RNA genetic regulators that control gene expression by altering their structures. The elucidation of riboswitch conformational changes before and after ligand recognition is crucial to understand how riboswitches can achieve high ligand binding affinity and discrimination against cellular analogs. The detailed characterization of riboswitch folding pathways suggest that they may use their intrinsic conformational dynamics to sample a large array of structures, some of which being nearly identical to ligand-bound molecules. Some of these structural conformers can be "captured" upon ligand binding, which is crucial for the outcome of gene regulation. Recent studies about the SAM-I riboswitch have revealed unexpected and previously unknown RNA folding mechanisms. For instance, the observed helical twist of the P1 stem upon ligand binding to the SAM-I aptamer adds a new element in the repertoire of RNA strategies for recognition of small metabolites. From an RNA folding perspective, these findings also strongly indicate that the SAM-I riboswitch could achieve ligand recognition by using an optimized combination of conformational capture and induced-fit approaches, a feature that may be shared by other RNA regulatory sequences.

PCNA and XPF cooperate to distort DNA substrates.

XPF is a structure-specific endonuclease that preferentially cleaves 3' DNA flaps during a variety of repair processes. The crystal structure of a crenarchaeal XPF protein bound to a DNA duplex yielded insights into how XPF might recognise branched DNA structures, and recent kinetic data have demonstrated that the sliding clamp PCNA acts as an essential cofactor, possibly by allowing XPF to distort the DNA structure into a proper conformation for efficient cleavage to occur. Here, we investigate the solution structure of the 3'-flap substrate bound to XPF in the presence and absence of PCNA using intramolecular Förster resonance energy transfer (FRET). We demonstrate that recognition of the flap substrate by XPF involves major conformational changes of the DNA, including a 90 degrees kink of the DNA duplex and organization of the single-stranded flap. In the presence of PCNA, there is a further substantial reorganization of the flap substrate bound to XPF, providing a structural basis for the observation that PCNA has an essential catalytic role in this system. The wider implications of these observations for the plethora of PCNA-dependent enzymes are discussed.

The influence of various regions of the FOXP2 sequence on its structure and DNA-binding function.

FOX proteins are a superfamily of transcription factors which share a DNA-binding domain referred to as the forkhead domain. Our focus is on the FOXP subfamily members, which are involved in language and cognition amongst other things. The FOXP proteins contain a conserved zinc finger and a leucine zipper motif in addition to the forkhead domain. The remainder of the sequence is predicted to be unstructured and includes an acidic C-terminal tail. In the present study, we aim to investigate how both the structured and unstructured regions of the sequence cooperate so as to enable FOXP proteins to perform their function. We do this by studying the effect of these regions on both oligomerisation and DNA binding. Structurally, the FOXP proteins appear to be comparatively globular with a high proportion of helical structure. The proteins multimerise via the leucine zipper, and the stability of the multimers is controlled by the unstructured interlinking sequence including the acid rich tail. FOXP2 is more compact than FOXP1, has a greater propensity to form higher order oligomers, and binds DNA with stronger affinity. We conclude that while the forkhead domain is necessary for DNA binding, the affinity of the binding event is attributable to the leucine zipper, and the unstructured regions play a significant role in the specificity of binding. The acid rich tail forms specific contacts with the forkhead domain which may influence oligomerisation and DNA binding, and therefore the acid rich tail may play an important regulatory role in FOXP transcription.

PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms: substrate targeting and catalytic step.

The sliding clamp Proliferating Cell Nuclear Antigen (PCNA) functions as a recruiter and organizer of a wide variety of DNA modifying enzymes including nucleases, helicases, polymerases and glycosylases. The 5'-flap endonuclease Fen-1 is essential for Okazaki fragment processing in eukaryotes and archaea, and is targeted to the replication fork by PCNA. Crenarchaeal XPF, a 3'-flap endonuclease, is also stimulated by PCNA in vitro. Using a novel continuous fluorimetric assay, we demonstrate that PCNA activates these two nucleases by fundamentally different mechanisms. PCNA stimulates Fen-1 by increasing the enzyme's binding affinity for substrates, as suggested previously. However, PCNA activates XPF by increasing the catalytic rate constant by four orders of magnitude without affecting the K(M). PCNA may function as a platform upon which XPF exerts force to distort DNA substrates, destabilizing the substrate and/or stabilizing the transition state structure. This suggests that PCNA can function directly in supporting catalysis as an essential cofactor in some circumstances, a new role for a protein that is generally assumed to perform a passive targeting and organizing function in molecular biology. This could provide a mechanism for the exquisite control of nuclease activity targeted to specific circumstances, such as replication forks or damaged DNA with pre-loaded PCNA.