Folding thermodynamics and kinetics of YNMG RNA hairpins: specific incorporation of 8-bromoguanosine leads to stabilization by enhancement of the folding rate.

Modified nucleotides allow fundamental energetic and kinetic properties of nucleic acids to be probed. Here, we demonstrate that an RNA hairpin containing the nucleotide analogue 8-bromoguanosine (8BrG or G), gcUUCGgc, has enhanced stability relative to the unmodified hairpin, with DeltaDeltaG(37)(degrees)= -0.69 +/- 0.15 kcal mol(-1) and DeltaT(M) = +6.8 +/- 1.4 degrees C. NMR spectroscopic data suggest that the enhanced stability of gcUUCGgc does not arise from the native state; laser temperature-jump experiments support this notion, as gcUUCGgc and gcUUCGgc have similar unfolding rate constants, but the folding rate constant of gcUUCGgc is 4.1-fold faster at 37.5 degrees C and 2.8-fold faster under isoenergetic conditions. On the basis of these findings, we propose that 8BrG reduces the conformational entropy of the denatured state, resulting in an accelerated conformational search for the native state and enhanced stability.

[1]  K. Hall,et al.  Experimental and computational studies of the G[UUCG]C RNA tetraloop. , 2000, Journal of molecular biology.

[2]  M. Gruebele,et al.  Laser Temperature Jump Induced Protein Refolding , 1998 .

[3]  Methinks it is like a folding curve. , 2002, Biophysical chemistry.

[4]  G. Fasman,et al.  Handbook of biochemistry and molecular biology. Nucleic acids - v. 1 - 3. ed. , 1975 .

[5]  J. Feigon,et al.  Solution structures of stem-loop RNAs that bind to the two N-terminal RNA-binding domains of nucleolin. , 2003, Nucleic acids research.

[6]  M. Ikehara,et al.  Studies on the conformation of purine nucleosides and their 5'-phosphates. , 1972, Biochemistry.

[7]  T. Pan,et al.  The thermodynamic origin of the stability of a thermophilic ribozyme , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[8]  I. Tinoco,et al.  Use of ultra stable UNCG tetraloop hairpins to fold RNA structures: thermodynamic and spectroscopic applications. , 1995, Nucleic acids research.

[9]  D. K. Treiber,et al.  An optimal Mg(2+) concentration for kinetic folding of the tetrahymena ribozyme. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[10]  T. Pan,et al.  Folding of a large ribozyme during transcription and the effect of the elongation factor NusA. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Philip C Bevilacqua,et al.  Isolation and characterization of a family of stable RNA tetraloops with the motif YNMG that participate in tertiary interactions. , 2002, Biochemistry.

[12]  K. Hall,et al.  Thermodynamics of 2'-ribose substitutions in UUCG tetraloops. , 2001, RNA.

[13]  H. M. Sobell,et al.  Crystal and molecular structure of 8-bromoguanosine and 8-bromoadenosine, two purine nucleosides in the syn conformation. , 1970, Journal of molecular biology.

[14]  I. Tinoco,et al.  Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops. , 1992, Nucleic acids research.

[15]  I. Tinoco,et al.  How RNA folds. , 1999, Journal of molecular biology.

[16]  Y. Mizutani,et al.  Nanosecond temperature jump and time-resolved Raman study of thermal unfolding of ribonuclease A. , 2000, Biophysical journal.

[17]  James R. Williamson,et al.  Chemical Probe for Glycosidic Conformation in Telomeric DNAs , 1994 .

[18]  D. Turner,et al.  Investigation of the structural basis for thermodynamic stabilities of tandem GU mismatches: solution structure of (rGAGGUCUC)2 by two-dimensional NMR and simulated annealing. , 1996, Biochemistry.

[19]  R. Kierzek,et al.  Restricting the conformational heterogeneity of RNA by specific incorporation of 8-bromoguanosine. , 2003, Journal of the American Chemical Society.

[20]  A. Michelson,et al.  Poly 8-bromoguanylic acid. , 1970, Biochimica et biophysica acta.

[21]  M. Gruebele,et al.  Observation of strange kinetics in protein folding. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[22]  R. Raines,et al.  Protein prosthesis: a nonnatural residue accelerates folding and increases stability. , 2003, Journal of the American Chemical Society.

[23]  D. K. Treiber,et al.  Beyond kinetic traps in RNA folding. , 2001, Current opinion in structural biology.

[24]  F. Eckstein,et al.  Dynamics of the RNA hairpin GNRA tetraloop. , 2000, Biochemistry.

[25]  D. Crothers,et al.  Nucleic Acids: Structures, Properties, and Functions , 2000 .

[26]  Ignacio Tinoco,et al.  Quantifying the energetic interplay of RNA tertiary and secondary structure interactions. , 1999, RNA.

[27]  D. Pörchke.  Molecular states in single‐stranded adenylate chains by relaxation analysis , 1978 .

[28]  A. J. Shaka,et al.  Evaluation of a new broadband decoupling sequence: WALTZ-16 , 1983 .

[29]  D. Draper,et al.  Bases defining an ammonium and magnesium ion-dependent tertiary structure within the large subunit ribosomal RNA. , 1994, Journal of molecular biology.

[30]  P. Bevilacqua,et al.  A mechanistic framework for co-transcriptional folding of the HDV genomic ribozyme in the presence of downstream sequence. , 2002, Journal of molecular biology.

[31]  D M Crothers,et al.  Kinetics of an RNA conformational switch. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[32]  C. A. Theimer,et al.  YNMG tetraloop formation by a dyskeratosis congenita mutation in human telomerase RNA. , 2003, RNA.

[33]  D. Turner,et al.  Laser Raman temperature-jump study of the kinetics of the triiodide equilibrium. Relaxation times in the 10-8 -10-7 second range , 1972 .

[34]  C. E. Longfellow,et al.  Laser temperature-jump, spectroscopic, and thermodynamic study of salt effects on duplex formation by dGCATGC. , 1989, Biochemistry.

[35]  K. Hall,et al.  Experimental and theoretical studies of the effects of deoxyribose substitutions on the stability of the UUCG tetraloop. , 2000, Journal of molecular biology.

[36]  C. Vonrhein,et al.  Structure of the 30S ribosomal subunit , 2000, Nature.

[37]  R. L. Baldwin,et al.  Are denatured proteins ever random coils? , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[38]  S. Neidle.  Topics in Nucleic Acid Structure , 1981 .

[39]  Teizo Kitagawa,et al.  Construction of Novel Nanosecond Temperature Jump Apparatuses Applicable to Raman Measurements and Direct Observation of Transient Temperature , 2000 .

[40]  R. Andino,et al.  Extending the family of UNCG-like tetraloop motifs: NMR structure of a CACG tetraloop from coxsackievirus B3. , 2003, Biochemistry.

[41]  I. Tinoco,et al.  Absorbance melting curves of RNA. , 1989, Methods in enzymology.

[42]  D. Turner,et al.  Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. , 1998, Biochemistry.

[43]  I. Tinoco,et al.  A thermodynamic study of unusually stable RNA and DNA hairpins. , 1991, Nucleic acids research.

[44]  G. Rose,et al.  A complete conformational map for RNA. , 1999, Journal of molecular biology.

[45]  A. Ansari,et al.  Loop Dependence of the Dynamics of DNA Hairpins , 2001 .

[46]  R. Srinivasan,et al.  A physical basis for protein secondary structure. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[47]  Shi-Jie Chen,et al.  RNA hairpin-folding kinetics , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[48]  T. Huynh-Dinh,et al.  UNCG tetraloops in short oligoribonucleotides reveal high thermodynamic stability and unusual structural properties in aqueous phase as confirmed by optical and NMR spectroscopies , 1997 .

[49]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[50]  M. Gruebele,et al.  Submicrosecond real-time fluorescence sampling: application to protein folding. , 2000, Journal of photochemistry and photobiology. B, Biology.

[51]  G. Varani,et al.  Structure of an unusually stable RNA hairpin. , 1991, Biochemistry.

[52]  D. Porschke,et al.  Model calculations on the kinetics of oligonucleotide double helix coil transitions. Evidence for a fast chain sliding reaction. , 1974 .

[53]  P. R. Bevington,et al.  Data Reduction and Error Analysis for the Physical Sciences , 1969 .

[54]  V. Pande,et al.  Absolute comparison of simulated and experimental protein-folding dynamics , 2002, Nature.

[55]  Daniel Herschlag,et al.  RNA simulations: probing hairpin unfolding and the dynamics of a GNRA tetraloop. , 2002, Journal of molecular biology.

[56]  A. Stein,et al.  Allosteric interpretation of Mg2+ binding to the denaturable Escherichia coli tRNAGlu2+. , 1976, Biochemistry.

[57]  J. Cavanagh.  Protein NMR Spectroscopy: Principles and Practice , 1995 .

[58]  Enrico Di Cera.  Thermodynamics in biology , 2000 .

[59]  C. Matthews,et al.  Probing the folding mechanism of a leucine zipper peptide by stopped-flow circular dichroism spectroscopy. , 1995, Biochemistry.

[60]  O. Bilsel,et al.  Folding Mechanism of the α-Subunit of Tryptophan Synthase, an α/β Barrel Protein: Global Analysis Highlights the Interconversion of Multiple Native, Intermediate, and Unfolded Forms through Parallel Channels† , 1999 .

[61]  Yiqing Shen,et al.  Configurational diffusion down a folding funnel describes the dynamics of DNA hairpins , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[62]  S. Balasubramanian,et al.  Non-Arrhenius kinetics for the loop closure of a DNA hairpin , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[63]  Martin Gruebele,et al.  Folding at the speed limit , 2003, Nature.

[64]  Arnold Weissberger,et al.  Investigation of Rates and Mechanisms of Reactions , 1974 .

[65]  R. Hartmann,et al.  Selection of Hammerhead Ribozyme Variants with Low Mg2+ Requirement: Importance of Stem‐Loop II , 2002, Chembiochem : a European journal of chemical biology.

[66]  S. Woodson,et al.  Effect of transcription on folding of the Tetrahymena ribozyme. , 2003, RNA.

[67]  C. Hervé du Penhoat,et al.  Common structural features of UUCG and UACG tetraloops in very short hairpins determined by UV absorption, Raman, IR and NMR spectroscopies. , 1997, Journal of biomolecular structure & dynamics.

[68]  D Thirumalai,et al.  Native secondary structure formation in RNA may be a slave to tertiary folding. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[69]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[70]  G. Stormo,et al.  CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[71]  T. Pan,et al.  Stepwise conversion of a mesophilic to a thermophilic ribozyme. , 2003, Journal of molecular biology.

[72]  Eric J. Sorin,et al.  Insights into nucleic acid conformational dynamics from massively parallel stochastic simulations. , 2003, Biophysical journal.

[73]  J. Puglisi,et al.  Biochemical and NMR studies of RNA conformation with an emphasis on RNA pseudoknots. , 1995, Methods in enzymology.

[74]  T. Huynh-Dinh,et al.  Structural features of the UCCG and UGCG tetraloops in very short hairpins as evidenced by optical spectroscopy. , 1998, Biochemistry.

[75]  I. Tinoco,et al.  RNA folding causes secondary structure rearrangement. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[76]  M. Sundaralingam,et al.  Probing the interrelation between the glycosyl torsion, sugar pucker, and the backbone conformation in C(8) substituted adenine nucleotides by 1H and 1H-(31P) fast Fourier transform nuclear magnetic resonance methods and conformational energy calculations. , 1974, Journal of the American Chemical Society.