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Folding Heterogeneity in HIV-1 Frameshifting Hairpin
368a
Tuesday, February 14, 2017
draw the T2 RNA back, the re-folded RNA must be unfolded prior to returning. Importantly, the detected unfolding time (stability marker) can clearly report the identity of each folding state, from the free chain to the hairpin, and to the final pseudoknot. The time-dependent occurrence probabilities of all the folding states help us to map the entire stepwise folding pathway in the time scale from 1 second to several minutes. This RNA single-molecule folding approach would lead to broad applications in detecting RNA structuredetermined biological functionalities and their regulation by ligands. 1809-Pos Board B129 Folding Heterogeneity in HIV-1 Frameshifting Hairpin Dustin B. Ritchie1, Collin Tittle1, Negar Rezajooei1, Logan Rouleau1, Tonia R. Cappellano1, William Sikkema1, Michael T. Woodside1,2. 1 Physics, University of Alberta, Edmonton, AB, Canada, 2National Institute for Nanotechnology, National Research Council, Edmonton, AB, Canada. Ribosomes translate mRNA in 3-nucleotide steps, maintaining an open reading frame until a stop codon is reached. Programmed 1 ribosomal frameshifting (PRF), whereby the ribosome is forced backward by 1 nt to shift the reading frame, is essential for propagation of many viruses. Frameshifting depends on two mRNA structures: a slippery sequence and a downstream stimulatory structure. PRF in HIV-1 is thought to be stimulated by a hairpin, although controversy around the role of a potential pseudoknot exists. The conformational dynamics of the stimulatory structure under tension applied by the ribosomal helicase during translation may play an important role in PRF, therefore we used optical tweezers to apply tension to the HIV-1 frameshift signal and monitor the folding and unfolding dynamics. The folding and unfolding kinetics and energy landscape of the hairpin were measured by ramping the force on the hairpin up and down, providing a detailed biophysical characterisation of the hairpin. Whereas the unfolding reflected the simple two-state behavior typical of many hairpins, unexpectedly, refolding was not just a reversal of the two-state unfolding, but instead displayed more heterogeneous kinetics. Evidence was found for multiple refolding pathways as well as previously unsuspected, partially folded intermediates. A longer HIV-1 frameshifting-signal construct with the potential to form the suspected pseudoknot was also measured. This construct typically displayed very similar mechanical properties to the hairpin alone, perhaps with more heterogeneous unfolding. Our observations of the HIV-1 frameshifting hairpin under tension suggest a possible functional role in PRF similar to the dynamics observed for frameshifting pseudoknots. 1810-Pos Board B130 Structural Insights to the 30 UTR of Gait Elements Nancy Wells1, Blanton S. Tolbert2. 1 Case Western Reserve, Cleveland Hts, OH, USA, 2Case Western Reserve, Cleveland, OH, USA. In the Interferon-Gamma-Activated Inhibitor of Translation (GAIT) system, a single inflammatory signal Interferon g (IFN-g) activates both transcriptional on and off-switches in human myeloid cells through the use of structured mRNAs. There is current data suggesting that these pro-inflammatory mRNAs may regulate the GAIT complex via structures in their 3’ UTRs. Several studies have demonstrated that these structured RNAs are capable of recognition and regulation of the GAIT system for multiple pro-inflammatory mRNAs and thus forming a post transcriptional regulon. At present there is no structural information on these GAIT elements. To understand the role that GAIT structure plays in regulating inflammation preliminary NMR experiments have been performed. The following four GAIT elements have been examined: Cp, CCL22, CCR4, and CXCL13. All four fold into thermodynamically stable stem loops despite sequence variation. Each RNA folds into stable stem loop structures containing an internal loop, apical hairpin loop, and stably defined upper and lower stem. Various temperature studies have revealed that the GAIT element for a stable helical region. 1811-Pos Board B131 Ligand-Directed Conformational Dynamics of the Adenine-Sensing Riboswitch Thermostat Sven Warhaut, Klara Rebecca Mertinkus, Philipp Ho¨llthaler, Boris F€urtig, Mike Heilemann, Martin Hengesbach, Harald Schwalbe. Goethe University Frankfurt, Frankfurt am Main, Germany. Bacterial gene expression can be regulated by translational operating riboswitches, cis-acting mRNA elements that exhibit ligand-dependent conformational switching between gene-OFF and gene-ON states with trapped or exposed ribosome binding site, respectively. The adenine-sensing riboswitch (Asw) located in the 5’ leader sequence of the add gene of the Gram-negative, human pathogenic, marine bacterium Vibrio vulnificus is the first discovered temperaturecompensated riboswitch, a riboswitch thermostat, that adjusts the relative populations of an adenine-binding competent and a binding incompetent apo conformation to compensate the inherent temperature-dependence of ligand binding. We have performed an integrated NMR and single-molecule FRET (smFRET) spec-
troscopic study of the conformational dynamics of this paradigmatic riboswitch with three stable macrostates. We demonstrate that NMR and smFRET revealed complementing folding transitions of the add Asw on the second to minute timescale for which we could map individual ligand-dependencies. Our results suggest that the adenine-sensing riboswitch thermostat operates via a ligand-induced equilibrium shift in persistent multi-state conformational dynamics. 1812-Pos Board B132 Dynamic Equilibrium of the TPP Riboswitch as Observed by MFD Fret Junyan Ma1, Soheila Rezaei2, Feng Ding2, Hugo Sanabria2. 1 Chemistry, Clemson University, Clemson, SC, USA, 2Physics and Astronomy, Clemson University, Clemson, SC, USA. Antibiotic-resistant infections are one of the widely known health problems that threaten over 2 million people each year in United State, and over 23,000 people died due to antibiotic resistant bacteria. Recent studies have proposed an innovative approach to fight antibiotic resistant bacteria using mRNA as targets for new potential drugs. Riboswitches are one of the most studied messenger RNAs that control gene function and respond to second messengers such as small molecules and proteins and could be used as targets. However, riboswitches are highly dynamic structures and evade most common methods of characterization. Using a structure-guided drug design rationale, our first goal is to determine the structure-function relationship of riboswitches upon binding of effector molecules. We utilize Fo¨rster Resonance Energy Transfer (FRET) at a single molecule level in Multiparameter Fluorescence Detection mode to understand the relationship between structure and dynamic of the TPP Riboswitch. We compare our results with Discrete Molecular Dynamic (DMD) simulations and find that the TPP-riboswitch is in equilibrium between two conformational states, which are potential targets to bind new small molecules. We alter the dynamic equilibrium by the presence TPP, MgCl2 and TPPþMgCl2. 1813-Pos Board B133 Folding and Catalysis of the Glms Ribozyme Riboswitch Studied at the Single-Molecule Level Andrew Savinov1, Steven M. Block2. 1 Biophysics Program, Stanford University, Stanford, CA, USA, 2 Departments of Applied Physics and Biology, Stanford University, Stanford, CA, USA. We present findings from optical trapping experiments performed on the glmS ribozyme riboswitch. Found in many Gram-positive bacteria, the glmS riboswitch down-regulates GlmS expression in the presence of the cell wall precursor—and enzymatic product of GlmS—glucosamine-6-phosphate (GlcN6P). In response to GlcN6P, the riboswitch site-specifically cleaves itself near its 50 end, which targets the glmS mRNA for subsequent degradation1. We performed self-cleavage assays on optically trapped ribozyme molecules in the presence of either GlcN6P or its catalytically inactive analog, Glc6P (glucose 6-phosphate). The assays demonstrate that our experimental construct, consisting of aminimal ribozyme sequence, is enzymatically active and strictly dependent upon GlcN6P. We also performed single-molecule force spectroscopy on the ribozyme catalytic core, measuring its unfolding/folding dynamics under external loads. The data reveal a series of intermediate folding states occupied during mechanically-induced denaturation and renaturation. Analysis of the data leads to a model for the folding pathway, involving sequential passage through a series of discrete substructures. We have measured kinetic and thermodynamic parameters for substructure formation, and are using these values to reconstruct a folding-energy landscape for the ribozyme. We are also determining the effect of cofactor binding on the folding-energy landscape, using the inactive analog Glc6P. Single-molecule self-cleavage activity measurements under a range of controlled loads allow us to determine the profile of catalytic activity in response to destabilizing forces. By combining results from measurements of folding and catalytic activity, we gain further insights into how specific structural features of this ribozyme relate to its catalytic function. Footnotes 1 Collins, J.A., Irnov, I., Baker, S., and Winkler, W.C. (2007) Genes Dev 21(24), 3356-68 1814-Pos Board B134 Mechanistic Insights into Functional Protein-RNA Interactions Involved in Viral Replication Blanton S. Tolbert. Case Western Reserve University, Cleveland, OH, USA. To replicate their genomes, viruses use conserved RNA structures that usurp host RNA binding proteins. The mechanisms by which viral RNA structure and sequence contribute to functional host interactions are still poorly understood. Here, we used High Throughput Sequencing Equilibrium (HTS-EQ) binding experiments combined with independent biophysical measurements to describe the complete affinity distribution of human hnRNP A1 bound to the HIV ESS3 stem
Tuesday, February 14, 2017
draw the T2 RNA back, the re-folded RNA must be unfolded prior to returning. Importantly, the detected unfolding time (stability marker) can clearly report the identity of each folding state, from the free chain to the hairpin, and to the final pseudoknot. The time-dependent occurrence probabilities of all the folding states help us to map the entire stepwise folding pathway in the time scale from 1 second to several minutes. This RNA single-molecule folding approach would lead to broad applications in detecting RNA structuredetermined biological functionalities and their regulation by ligands. 1809-Pos Board B129 Folding Heterogeneity in HIV-1 Frameshifting Hairpin Dustin B. Ritchie1, Collin Tittle1, Negar Rezajooei1, Logan Rouleau1, Tonia R. Cappellano1, William Sikkema1, Michael T. Woodside1,2. 1 Physics, University of Alberta, Edmonton, AB, Canada, 2National Institute for Nanotechnology, National Research Council, Edmonton, AB, Canada. Ribosomes translate mRNA in 3-nucleotide steps, maintaining an open reading frame until a stop codon is reached. Programmed 1 ribosomal frameshifting (PRF), whereby the ribosome is forced backward by 1 nt to shift the reading frame, is essential for propagation of many viruses. Frameshifting depends on two mRNA structures: a slippery sequence and a downstream stimulatory structure. PRF in HIV-1 is thought to be stimulated by a hairpin, although controversy around the role of a potential pseudoknot exists. The conformational dynamics of the stimulatory structure under tension applied by the ribosomal helicase during translation may play an important role in PRF, therefore we used optical tweezers to apply tension to the HIV-1 frameshift signal and monitor the folding and unfolding dynamics. The folding and unfolding kinetics and energy landscape of the hairpin were measured by ramping the force on the hairpin up and down, providing a detailed biophysical characterisation of the hairpin. Whereas the unfolding reflected the simple two-state behavior typical of many hairpins, unexpectedly, refolding was not just a reversal of the two-state unfolding, but instead displayed more heterogeneous kinetics. Evidence was found for multiple refolding pathways as well as previously unsuspected, partially folded intermediates. A longer HIV-1 frameshifting-signal construct with the potential to form the suspected pseudoknot was also measured. This construct typically displayed very similar mechanical properties to the hairpin alone, perhaps with more heterogeneous unfolding. Our observations of the HIV-1 frameshifting hairpin under tension suggest a possible functional role in PRF similar to the dynamics observed for frameshifting pseudoknots. 1810-Pos Board B130 Structural Insights to the 30 UTR of Gait Elements Nancy Wells1, Blanton S. Tolbert2. 1 Case Western Reserve, Cleveland Hts, OH, USA, 2Case Western Reserve, Cleveland, OH, USA. In the Interferon-Gamma-Activated Inhibitor of Translation (GAIT) system, a single inflammatory signal Interferon g (IFN-g) activates both transcriptional on and off-switches in human myeloid cells through the use of structured mRNAs. There is current data suggesting that these pro-inflammatory mRNAs may regulate the GAIT complex via structures in their 3’ UTRs. Several studies have demonstrated that these structured RNAs are capable of recognition and regulation of the GAIT system for multiple pro-inflammatory mRNAs and thus forming a post transcriptional regulon. At present there is no structural information on these GAIT elements. To understand the role that GAIT structure plays in regulating inflammation preliminary NMR experiments have been performed. The following four GAIT elements have been examined: Cp, CCL22, CCR4, and CXCL13. All four fold into thermodynamically stable stem loops despite sequence variation. Each RNA folds into stable stem loop structures containing an internal loop, apical hairpin loop, and stably defined upper and lower stem. Various temperature studies have revealed that the GAIT element for a stable helical region. 1811-Pos Board B131 Ligand-Directed Conformational Dynamics of the Adenine-Sensing Riboswitch Thermostat Sven Warhaut, Klara Rebecca Mertinkus, Philipp Ho¨llthaler, Boris F€urtig, Mike Heilemann, Martin Hengesbach, Harald Schwalbe. Goethe University Frankfurt, Frankfurt am Main, Germany. Bacterial gene expression can be regulated by translational operating riboswitches, cis-acting mRNA elements that exhibit ligand-dependent conformational switching between gene-OFF and gene-ON states with trapped or exposed ribosome binding site, respectively. The adenine-sensing riboswitch (Asw) located in the 5’ leader sequence of the add gene of the Gram-negative, human pathogenic, marine bacterium Vibrio vulnificus is the first discovered temperaturecompensated riboswitch, a riboswitch thermostat, that adjusts the relative populations of an adenine-binding competent and a binding incompetent apo conformation to compensate the inherent temperature-dependence of ligand binding. We have performed an integrated NMR and single-molecule FRET (smFRET) spec-
troscopic study of the conformational dynamics of this paradigmatic riboswitch with three stable macrostates. We demonstrate that NMR and smFRET revealed complementing folding transitions of the add Asw on the second to minute timescale for which we could map individual ligand-dependencies. Our results suggest that the adenine-sensing riboswitch thermostat operates via a ligand-induced equilibrium shift in persistent multi-state conformational dynamics. 1812-Pos Board B132 Dynamic Equilibrium of the TPP Riboswitch as Observed by MFD Fret Junyan Ma1, Soheila Rezaei2, Feng Ding2, Hugo Sanabria2. 1 Chemistry, Clemson University, Clemson, SC, USA, 2Physics and Astronomy, Clemson University, Clemson, SC, USA. Antibiotic-resistant infections are one of the widely known health problems that threaten over 2 million people each year in United State, and over 23,000 people died due to antibiotic resistant bacteria. Recent studies have proposed an innovative approach to fight antibiotic resistant bacteria using mRNA as targets for new potential drugs. Riboswitches are one of the most studied messenger RNAs that control gene function and respond to second messengers such as small molecules and proteins and could be used as targets. However, riboswitches are highly dynamic structures and evade most common methods of characterization. Using a structure-guided drug design rationale, our first goal is to determine the structure-function relationship of riboswitches upon binding of effector molecules. We utilize Fo¨rster Resonance Energy Transfer (FRET) at a single molecule level in Multiparameter Fluorescence Detection mode to understand the relationship between structure and dynamic of the TPP Riboswitch. We compare our results with Discrete Molecular Dynamic (DMD) simulations and find that the TPP-riboswitch is in equilibrium between two conformational states, which are potential targets to bind new small molecules. We alter the dynamic equilibrium by the presence TPP, MgCl2 and TPPþMgCl2. 1813-Pos Board B133 Folding and Catalysis of the Glms Ribozyme Riboswitch Studied at the Single-Molecule Level Andrew Savinov1, Steven M. Block2. 1 Biophysics Program, Stanford University, Stanford, CA, USA, 2 Departments of Applied Physics and Biology, Stanford University, Stanford, CA, USA. We present findings from optical trapping experiments performed on the glmS ribozyme riboswitch. Found in many Gram-positive bacteria, the glmS riboswitch down-regulates GlmS expression in the presence of the cell wall precursor—and enzymatic product of GlmS—glucosamine-6-phosphate (GlcN6P). In response to GlcN6P, the riboswitch site-specifically cleaves itself near its 50 end, which targets the glmS mRNA for subsequent degradation1. We performed self-cleavage assays on optically trapped ribozyme molecules in the presence of either GlcN6P or its catalytically inactive analog, Glc6P (glucose 6-phosphate). The assays demonstrate that our experimental construct, consisting of aminimal ribozyme sequence, is enzymatically active and strictly dependent upon GlcN6P. We also performed single-molecule force spectroscopy on the ribozyme catalytic core, measuring its unfolding/folding dynamics under external loads. The data reveal a series of intermediate folding states occupied during mechanically-induced denaturation and renaturation. Analysis of the data leads to a model for the folding pathway, involving sequential passage through a series of discrete substructures. We have measured kinetic and thermodynamic parameters for substructure formation, and are using these values to reconstruct a folding-energy landscape for the ribozyme. We are also determining the effect of cofactor binding on the folding-energy landscape, using the inactive analog Glc6P. Single-molecule self-cleavage activity measurements under a range of controlled loads allow us to determine the profile of catalytic activity in response to destabilizing forces. By combining results from measurements of folding and catalytic activity, we gain further insights into how specific structural features of this ribozyme relate to its catalytic function. Footnotes 1 Collins, J.A., Irnov, I., Baker, S., and Winkler, W.C. (2007) Genes Dev 21(24), 3356-68 1814-Pos Board B134 Mechanistic Insights into Functional Protein-RNA Interactions Involved in Viral Replication Blanton S. Tolbert. Case Western Reserve University, Cleveland, OH, USA. To replicate their genomes, viruses use conserved RNA structures that usurp host RNA binding proteins. The mechanisms by which viral RNA structure and sequence contribute to functional host interactions are still poorly understood. Here, we used High Throughput Sequencing Equilibrium (HTS-EQ) binding experiments combined with independent biophysical measurements to describe the complete affinity distribution of human hnRNP A1 bound to the HIV ESS3 stem