Tuning riboswitch regulation through conformational selection.

The S(MK) box riboswitch, which represents one of three known classes of S-adenosylmethionine (SAM)-responsive riboswitches, regulates gene expression in bacteria at the level of translation initiation. In contrast to most riboswitches, which contain separate domains responsible for ligand recognition and gene regulation, the ligand-binding and regulatory domains of the S(MK) box riboswitch are coincident. This property was exploited to allow the first atomic-level characterization of a functionally intact riboswitch in both the ligand-bound state and the ligand-free state. NMR spectroscopy revealed distinct mutually exclusive RNA conformations that are differentially populated in the presence or in the absence of the effector metabolite. Isothermal titration calorimetry and in vivo reporter assay results revealed the thermodynamic and functional consequences of this conformational equilibrium. We present a comprehensive model of the structural, thermodynamic, and functional properties of this compact RNA regulatory element.

[1]  Peter V. Konarev,et al.  MASSHA – a graphics system for rigid-body modelling of macromolecular complexes against solution scattering data , 2001 .

[2]  R. Batey,et al.  Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain. , 2006, Journal of molecular biology.

[3]  F. D’Anselmi,et al.  Evaluation of chemical and diastereoisomeric stability of S-adenosylmethionine in aqueous solution by capillary electrophoresis. , 2005, Journal of pharmaceutical and biomedical analysis.

[4]  John C. Lindon,et al.  Improved WATERGATE Pulse Sequences for Solvent Suppression in NMR Spectroscopy , 1998 .

[5]  Dmitri I. Svergun,et al.  Automated matching of high- and low-resolution structural models , 2001 .

[6]  A. Serganov,et al.  Structural insights into amino acid binding and gene control by a lysine riboswitch , 2008, Nature.

[7]  Dmitri I. Svergun,et al.  PRIMUS: a Windows PC-based system for small-angle scattering data analysis , 2003 .

[8]  Dmitri I. Svergun,et al.  Uniqueness of ab initio shape determination in small-angle scattering , 2003 .

[9]  H. Heus,et al.  A network of heterogeneous hydrogen bonds in GNRA tetraloops. , 1996, Journal of molecular biology.

[10]  T. Henkin,et al.  S-adenosylmethionine directly inhibits binding of 30S ribosomal subunits to the SMK box translational riboswitch RNA , 2007, Proceedings of the National Academy of Sciences.

[11]  S. Grzesiek,et al.  Direct detection of N−H⋯O=C hydrogen bonds in biomolecules by NMR spectroscopy , 2008, Nature Protocols.

[12]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[13]  T. Henkin,et al.  The SMK box is a new SAM-binding RNA for translational regulation of SAM synthetase , 2006, Nature Structural &Molecular Biology.

[14]  A. Pardi,et al.  Role of a heterogeneous free state in the formation of a specific RNA-theophylline complex. , 2003, Biochemistry.

[15]  L. W. Parks,et al.  The stability and hydrolysis of S-adenosylmethionine; isolation of S-ribosylmethionine. , 1958, The Journal of biological chemistry.

[16]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[17]  R. Batey,et al.  Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element* , 2008, Journal of Biological Chemistry.

[18]  T. Henkin,et al.  Crystal structures of the SAM-III/SMK riboswitch reveal the SAM-dependent translation inhibition mechanism , 2008, Nature Structural &Molecular Biology.

[19]  R. Montange,et al.  Free state conformational sampling of the SAM-I riboswitch aptamer domain. , 2010, Structure.

[20]  R. Batey,et al.  Structure of the SAM-II riboswitch bound to S-adenosylmethionine , 2008, Nature Structural &Molecular Biology.

[21]  I. Tinoco,et al.  Solution structure of Cobalt(III)hexammine complexed to the GAAA tetraloop, and metal-ion binding to G.A mismatches. , 2000, Journal of molecular biology.

[22]  E. R. Allen,et al.  Genetic mapping and physiological consequences of metE mutations of Bacillus subtilis , 1988, Journal of bacteriology.

[23]  D I Svergun,et al.  Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. , 1999, Biophysical journal.

[24]  Harald Schwalbe,et al.  Interplay of ‘induced fit’ and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch , 2006, Nucleic acids research.

[25]  D. Svergun,et al.  Synchrotron radiation solution X-ray scattering study of the pH dependence of the quaternary structure of yeast pyruvate decarboxylase. , 1992, Biochemistry.

[26]  A. Serganov,et al.  Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch , 2006, Nature.

[27]  Tina M. Henkin,et al.  Natural Variability in S-Adenosylmethionine (SAM)-Dependent Riboswitches: S-Box Elements in Bacillus subtilis Exhibit Differential Sensitivity to SAM In Vivo and In Vitro , 2007, Journal of bacteriology.

[28]  T. Henkin,et al.  tRNA as a positive regulator of transcription antitermination in B. subtilis , 1993, Cell.

[29]  T. Henkin,et al.  Riboswitch RNAs: Regulation of gene expression by direct monitoring of a physiological signal , 2010, RNA biology.

[30]  Ryan T Fuchs,et al.  The SAM‐responsive SMK box is a reversible riboswitch , 2010, Molecular microbiology.

[31]  T. Henkin,et al.  The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in Gram‐positive bacteria , 1998, Molecular microbiology.

[32]  S. Wijmenga,et al.  Ligand-induced folding of the guanine-sensing riboswitch is controlled by a combined predetermined induced fit mechanism. , 2007, RNA.

[33]  R. Montange,et al.  Structure of the S-adenosylmethionine riboswitch regulatory mRNA element , 2006, Nature.

[34]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[35]  T. Henkin,et al.  Transcription termination control of the S box system: Direct measurement of S-adenosylmethionine by the leader RNA , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[36]  C. Anagnostopoulos,et al.  REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS , 1961, Journal of bacteriology.

[37]  T. Henkin.  Riboswitch RNAs: using RNA to sense cellular metabolism. , 2008, Genes & development.

[38]  S. Grzesiek,et al.  Direct detection of N−H⋯N hydrogen bonds in biomolecules by NMR spectroscopy , 2008, Nature Protocols.

[39]  Jeffrey E. Barrick,et al.  Metabolite-binding RNA domains are present in the genes of eukaryotes. , 2003, RNA.

[40]  G. Fournet,et al.  Small‐Angle Scattering of X‐Rays , 1956 .

[41]  Jeffrey E. Barrick,et al.  Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria , 2005, Genome Biology.

[42]  Juan Miranda-Ríos,et al.  The THI-box riboswitch, or how RNA binds thiamin pyrophosphate. , 2007, Structure.

[43]  H. Schwalbe,et al.  Time-resolved NMR methods resolving ligand-induced RNA folding at atomic resolution , 2007, Proceedings of the National Academy of Sciences.

[44]  M R Eftink,et al.  Enthalpy-entropy compensation and heat capacity changes for protein-ligand interactions: general thermodynamic models and data for the binding of nucleotides to ribonuclease A. , 1983, Biochemistry.

[45]  J. Changeux,et al.  ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. , 1965, Journal of molecular biology.

[46]  Renate Rieder,et al.  Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach , 2007, Nucleic acids research.

[47]  R. Batey,et al.  A switch in time: detailing the life of a riboswitch. , 2009, Biochimica et biophysica acta.

[48]  Bruce A Johnson,et al.  Using NMRView to visualize and analyze the NMR spectra of macromolecules. , 2004, Methods in molecular biology.