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Folding and Catalysis of the glmS Ribozyme Riboswitch Studied at the Single-Molecule Level
410a
Tuesday, March 1, 2016
multiple conformational states.1 Furthermore, these studies showed that they have pronounced thermodynamic stability due to noncanonical interactions.2 Aiming to establish the p-T stability diagram and correlate it with theoretical predictions, UV-vis, Fourier-transform infrared (FTIR) and fluorescence resonance energy transfer (FRET) experiments were carried out over a wide range of temperatures and pressures. The combined results reveal characteristic conformational changes as a function of temperature and pressure. The thermal melting analysis revealed a broad, non-two-state melting transition between 40 and 60oC. Combined high-pressure FRET and UV results indicate that below the melting temperature pressure perturbs stem interactions and increases the population of non-native conformations. The high-pressure FTIR data showed that at ambient temperature, pressure is able to destabilize native stem interactions, without leading to complete unfolding, however. At high temperatures, e.g. 70 oC, in the unfolded state, pressure does not lead to significant refolding. The combined structural analysis seems to be compatible with a non-two-state elliptically shaped p-T stability diagram. (1)Garcia A. E.; Paschek D.J.; Am. Chem. Soc. 2008, 130, 815-817. (2)Chakraborty D., et al.; J. Am. Chem. Soc. 2014, 136, 18052-18061. Corresponding author: [email protected]. 2027-Pos Board B171 VfoldCPX Server for RNA/RNA Complex Structure Prediction Xiaojun Xu, Shi-Jie Chen. Physics and Astronomy, University of Missouri-Columbia, Columbia, MO, USA. RNA/RNA interactions are essential for genomic RNA dimerization and regulation of gene expression. Predicting the structure and folding stabilities of RNA/RNA complexes is a biologically significant problem. We recently developed a new web server, VfoldCPX, to predict two-dimensional (2D) structures of RNA/RNA complexes from the sequence. The server is based on a new Vfold model that employs coarse-grained entropy parameters for the different structural motifs. By taking into account both the intra- and inter-molecular interactions, the model can predict RNA/RNA structures with cross-linked tertiary contacts. Furthermore, in addition to the minimum free energy structure, the server can predict multiple energetically stable structures ranked by their stabilities. The predicted structures can offer users with extensive insights into the folding and conformational switches in RNA biological functions. The web server is freely accessible at ‘‘http://rna.physics.missouri.edu’’. 2028-Pos Board B172 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 the glmS gene in many 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 5´ end, which targets the mRNA for subsequent degradation1. We performed single-molecule force spectroscopy experiments on the ribozyme catalytic core, unfolding and refolding the glmS RNA under controlled mechanical loads. The force-extension curves (FECs) reveal that folding and unfolding occur through a series of intermediate states, and we observe a striking hysteresis between unfolding and refolding of the ribozyme. Analysis of the FEC data leads to a model for the major unfolding pathway, involving the sequential unfolding of a series of discrete substructures. We’ve also begun to characterize the folding energy landscape of the catalytic core, using a series of constantforce measurements. We performed self-cleavage assays with optically trapped ribozyme molecules in the presence of the enzymatic cofactor GlcN6P and its catalytically inactive analog, glucose 6-phosphate. These assays demonstrate that our experimental construct, consisting of aminimal ribozyme sequence, is enzymatically active and strictly dependent upon the presence of GlcN6P. Self-cleavage measurements under controlled loads allow us to determine the profile of the catalytic activity in response to destabilizing forces. We observe an intermediate destabilization regime where the ribozyme remains active, but displays a partial loss of function. Footnotes 1 Collins, J.A., Irnov, I., Baker, S., and Winkler, W.C. (2007) Genes Dev21(24), 3356-68.
2029-Pos Board B173 Kinetic Model of Mg2D Induced RNA Tertiary Folding from Stopped Flow Fluorescence Data Robb Welty, Michael J. Rau, Kathleen B. Hall. Biochem & Mol. Biophys, Washington University, St. Louis, MO, USA. Many of the biological processes that involve RNA rely on its ability to form complex tertiary structures. The process of how these molecules fold into these tertiary structures is not well understood, in part due to the unpredictability and complexity of the interactions among nucleotides that define the tertiary structures. To better understand RNA folding we are investigating the prokaryotic rRNA GTPase center, a 60 nucleotide RNA which folds in to a complex tertiary structure in the presence of Mg2þ ions. Its tertiary interactions include formation of base triples, a triloop, as well as long-range nucleobase stacking interactions. To monitor its folding process we have replaced key adenine residues with the fluorescent base analog 2-aminopurine in different positions of this RNA to probe distinct local environments. Stopped flow kinetics techniques were used to examine the coordination of these different elements in the context of global folding. Data from the stopped flow experiments were used to refine a theoretical model of the folding pathway of this RNA. Our model consists of four temporally resolved kinetic steps with approximate rate constants. This work was funded by the NIH R01-GM098102 to KBH. Labeled RNA molecules were contributed by Agilent. 2030-Pos Board B174 Crystal Structure of Group II Intron Domain 1 Reveals a Template for RNA Assembly Chen Zhao1, Marco Marcia2, Kanagalaghatta R. Rajashankar3, Anna Marie Pyle1. 1 Yale University, New Haven, CT, USA, 2EMBL, Grenoble, France, 3 Argonne National Laboratory, Lemont, IL, USA. Although many non-coding RNAs can form distinct three dimensional structures, our knowledge on how these structures are assembled is limited. This limitation is partly due to the lack of structural information on RNA folding intermediates. Here we report crystal structures of an obligate group II intron ˚ . This folding intermediate folding intermediate, solved at a resolution of 3 A is the first domain (D1) from the Oceanobacillus iheyensis (O.i.) group II intron, which has been shown to fold first and to serve as a scaffold, or template, for proper assembly of all other intron domains (1-3). When compared to D1 in the full-length intron (D1full) (4, 5), the isolated D1 (D1iso) adopts a native-like overall configuration, but its active-site binding cleft is closed. Comparative analysis of the backbone torsion angles reveals hinge motions that mediate the transition between the closed state as in D1iso and the open state as in D1full. Furthermore, B-factor analysis shows the central 5-way junction is the critical structural motif for dictating the shape of the native-like configuration. As D1 is both the first domain and a templating intermediate for folding of group II introns, out results provide structural evidence that a ‘‘first comes, first folds’’ strategy is possible, and they show how this strategy can facilitate a highly precise folding pathway for a large multi-domain RNA. 1. Pyle AM, Fedorova O, Waldsich C. 2007. Trends Biochem Sci 32: 138-45. 2. Su LJ, Waldsich C, Pyle AM. 2005. Nucleic Acids Res 33: 6674-87. 3. Qin PZ, Pyle AM. 1997. Biochemistry 36: 4718-30. 4. Toor N, Keating KS, Taylor SD, Pyle AM. 2008. Science 320: 77-82. 5. Marcia M, Pyle AM. 2012. Cell 151: 497-507. 2031-Pos Board B175 Thermodynamic Stabilities of Multibranch Loops in Bacteriophage Packaging RNA Alyssa Hill1, Susan Schroeder1,2. 1 Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, USA, 2Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA. Multibranch loops are key determinants of structural and functional roles in RNAs. In prohead RNA (pRNA), an essential component of the F29-like bacteriophage packaging motor, the three-way junction (3WJ) is a multibranch loop that imparts flexibility on the RNA, correctly placing helices in the spatial orientation necessary for in vivo self-assembly and packaging. In vitro, pRNA self-assembles, and some species are capable of forming dimers, trimers, and thermostable higher-order multimers. Although different pRNAs have very different interlocking loop sequences and thereby different base pairing stabilities, the overall energetics for pRNA self-assembly are conserved. We present thermodynamic data that support the hypothesis that 3WJ stabilities counterbalance loop-loop interaction stabilities in pRNA.
Tuesday, March 1, 2016
multiple conformational states.1 Furthermore, these studies showed that they have pronounced thermodynamic stability due to noncanonical interactions.2 Aiming to establish the p-T stability diagram and correlate it with theoretical predictions, UV-vis, Fourier-transform infrared (FTIR) and fluorescence resonance energy transfer (FRET) experiments were carried out over a wide range of temperatures and pressures. The combined results reveal characteristic conformational changes as a function of temperature and pressure. The thermal melting analysis revealed a broad, non-two-state melting transition between 40 and 60oC. Combined high-pressure FRET and UV results indicate that below the melting temperature pressure perturbs stem interactions and increases the population of non-native conformations. The high-pressure FTIR data showed that at ambient temperature, pressure is able to destabilize native stem interactions, without leading to complete unfolding, however. At high temperatures, e.g. 70 oC, in the unfolded state, pressure does not lead to significant refolding. The combined structural analysis seems to be compatible with a non-two-state elliptically shaped p-T stability diagram. (1)Garcia A. E.; Paschek D.J.; Am. Chem. Soc. 2008, 130, 815-817. (2)Chakraborty D., et al.; J. Am. Chem. Soc. 2014, 136, 18052-18061. Corresponding author: [email protected]. 2027-Pos Board B171 VfoldCPX Server for RNA/RNA Complex Structure Prediction Xiaojun Xu, Shi-Jie Chen. Physics and Astronomy, University of Missouri-Columbia, Columbia, MO, USA. RNA/RNA interactions are essential for genomic RNA dimerization and regulation of gene expression. Predicting the structure and folding stabilities of RNA/RNA complexes is a biologically significant problem. We recently developed a new web server, VfoldCPX, to predict two-dimensional (2D) structures of RNA/RNA complexes from the sequence. The server is based on a new Vfold model that employs coarse-grained entropy parameters for the different structural motifs. By taking into account both the intra- and inter-molecular interactions, the model can predict RNA/RNA structures with cross-linked tertiary contacts. Furthermore, in addition to the minimum free energy structure, the server can predict multiple energetically stable structures ranked by their stabilities. The predicted structures can offer users with extensive insights into the folding and conformational switches in RNA biological functions. The web server is freely accessible at ‘‘http://rna.physics.missouri.edu’’. 2028-Pos Board B172 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 the glmS gene in many 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 5´ end, which targets the mRNA for subsequent degradation1. We performed single-molecule force spectroscopy experiments on the ribozyme catalytic core, unfolding and refolding the glmS RNA under controlled mechanical loads. The force-extension curves (FECs) reveal that folding and unfolding occur through a series of intermediate states, and we observe a striking hysteresis between unfolding and refolding of the ribozyme. Analysis of the FEC data leads to a model for the major unfolding pathway, involving the sequential unfolding of a series of discrete substructures. We’ve also begun to characterize the folding energy landscape of the catalytic core, using a series of constantforce measurements. We performed self-cleavage assays with optically trapped ribozyme molecules in the presence of the enzymatic cofactor GlcN6P and its catalytically inactive analog, glucose 6-phosphate. These assays demonstrate that our experimental construct, consisting of aminimal ribozyme sequence, is enzymatically active and strictly dependent upon the presence of GlcN6P. Self-cleavage measurements under controlled loads allow us to determine the profile of the catalytic activity in response to destabilizing forces. We observe an intermediate destabilization regime where the ribozyme remains active, but displays a partial loss of function. Footnotes 1 Collins, J.A., Irnov, I., Baker, S., and Winkler, W.C. (2007) Genes Dev21(24), 3356-68.
2029-Pos Board B173 Kinetic Model of Mg2D Induced RNA Tertiary Folding from Stopped Flow Fluorescence Data Robb Welty, Michael J. Rau, Kathleen B. Hall. Biochem & Mol. Biophys, Washington University, St. Louis, MO, USA. Many of the biological processes that involve RNA rely on its ability to form complex tertiary structures. The process of how these molecules fold into these tertiary structures is not well understood, in part due to the unpredictability and complexity of the interactions among nucleotides that define the tertiary structures. To better understand RNA folding we are investigating the prokaryotic rRNA GTPase center, a 60 nucleotide RNA which folds in to a complex tertiary structure in the presence of Mg2þ ions. Its tertiary interactions include formation of base triples, a triloop, as well as long-range nucleobase stacking interactions. To monitor its folding process we have replaced key adenine residues with the fluorescent base analog 2-aminopurine in different positions of this RNA to probe distinct local environments. Stopped flow kinetics techniques were used to examine the coordination of these different elements in the context of global folding. Data from the stopped flow experiments were used to refine a theoretical model of the folding pathway of this RNA. Our model consists of four temporally resolved kinetic steps with approximate rate constants. This work was funded by the NIH R01-GM098102 to KBH. Labeled RNA molecules were contributed by Agilent. 2030-Pos Board B174 Crystal Structure of Group II Intron Domain 1 Reveals a Template for RNA Assembly Chen Zhao1, Marco Marcia2, Kanagalaghatta R. Rajashankar3, Anna Marie Pyle1. 1 Yale University, New Haven, CT, USA, 2EMBL, Grenoble, France, 3 Argonne National Laboratory, Lemont, IL, USA. Although many non-coding RNAs can form distinct three dimensional structures, our knowledge on how these structures are assembled is limited. This limitation is partly due to the lack of structural information on RNA folding intermediates. Here we report crystal structures of an obligate group II intron ˚ . This folding intermediate folding intermediate, solved at a resolution of 3 A is the first domain (D1) from the Oceanobacillus iheyensis (O.i.) group II intron, which has been shown to fold first and to serve as a scaffold, or template, for proper assembly of all other intron domains (1-3). When compared to D1 in the full-length intron (D1full) (4, 5), the isolated D1 (D1iso) adopts a native-like overall configuration, but its active-site binding cleft is closed. Comparative analysis of the backbone torsion angles reveals hinge motions that mediate the transition between the closed state as in D1iso and the open state as in D1full. Furthermore, B-factor analysis shows the central 5-way junction is the critical structural motif for dictating the shape of the native-like configuration. As D1 is both the first domain and a templating intermediate for folding of group II introns, out results provide structural evidence that a ‘‘first comes, first folds’’ strategy is possible, and they show how this strategy can facilitate a highly precise folding pathway for a large multi-domain RNA. 1. Pyle AM, Fedorova O, Waldsich C. 2007. Trends Biochem Sci 32: 138-45. 2. Su LJ, Waldsich C, Pyle AM. 2005. Nucleic Acids Res 33: 6674-87. 3. Qin PZ, Pyle AM. 1997. Biochemistry 36: 4718-30. 4. Toor N, Keating KS, Taylor SD, Pyle AM. 2008. Science 320: 77-82. 5. Marcia M, Pyle AM. 2012. Cell 151: 497-507. 2031-Pos Board B175 Thermodynamic Stabilities of Multibranch Loops in Bacteriophage Packaging RNA Alyssa Hill1, Susan Schroeder1,2. 1 Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, USA, 2Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA. Multibranch loops are key determinants of structural and functional roles in RNAs. In prohead RNA (pRNA), an essential component of the F29-like bacteriophage packaging motor, the three-way junction (3WJ) is a multibranch loop that imparts flexibility on the RNA, correctly placing helices in the spatial orientation necessary for in vivo self-assembly and packaging. In vitro, pRNA self-assembles, and some species are capable of forming dimers, trimers, and thermostable higher-order multimers. Although different pRNAs have very different interlocking loop sequences and thereby different base pairing stabilities, the overall energetics for pRNA self-assembly are conserved. We present thermodynamic data that support the hypothesis that 3WJ stabilities counterbalance loop-loop interaction stabilities in pRNA.