Studies of the Molecular Origins of Thermodynamic Stability in RNA Secondary Structure

Open Access
Proctor, David James
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
May 04, 2004
Committee Members:
  • Philip C. Bevilacqua, Committee Chair
  • Juliette T J Lecomte, Committee Member
  • George I Makhatadze, Committee Member
  • Paul Lee Babitzke, Committee Member
  • RNA
  • biophysics
  • structure
  • thermodynamics
Ribonucleic acid is central to numerous cellular events including transcription, translation, gene regulation, and cell signaling. The biological function of a given RNA often depends on the formation of a distinctive tertiary structure. The formation of tertiary structure typically develops in a hierarchical fashion using a variety of aromatic stacking and hydrogen bonding interactions, or secondary structural motifs, as building blocks. In addition to the role of secondary structure in RNA function and interaction in vivo, the thermodynamic stability of secondary structural motifs is critical for tertiary structure formation. This thesis describes fundamental studies of the molecular basis for thermodynamic stability in RNA secondary structure. Hairpins are the dominant secondary structure in RNA, and are known to function in nucleating RNA folding, RNA-RNA tertiary interactions, and RNA-protein interactions. The stability and structure of RNA tetraloop hairpins have been studied extensively, with thermodynamic and phylogenetic studies revealing that the majority of conserved tetraloop-closing base pair combinations are exceptionally stable. This suggests that thermodynamically stable RNA secondary structures may serve important biological roles. A family of sequences having the motif YNMG (Y = C or U; N = A, C, G, or U; M = A or C) was isolated from a combinatorial library of 4096 sequences using in vitro selection in combination with temperature gradient gel electrophoresis. Thermodynamic analysis of YNMG and variant tetraloops confirmed optimal stability with Y at tetraloop position 1 and M at position 3. Similarity in structure and stability among YNMG hairpins was further supported by deoxyribose substitution, CD, and NMR experiments. Taken together, these results support the existence of a family of UNCG-like tetraloops with the motif YNMG that are thermodynamically stable and structurally similar. Structural similarity among hairpin tetraloops having the motif YNMG has been independently verified by determination of the solution structures of the CACG, CCCG, and UCAGU tetraloops in other research laboratories. Moreover, an electrostatic origin for the importance of the closing base pair in imparting thermodynamic stability was determined in the context of these structures. Finally, phylogenetic and structural data suggested that YNAG tetraloops in particular have enhanced flexibility, which allows tertiary interactions to be maintained in the context of diverse loop sequences and illustrates the relationship between thermodynamic stability and loop flexibility. Native, functional RNA secondary and tertiary structure develops from a random ensemble of states through discrete folding pathways, or mechanisms. For nucleic acids, the simplicity of rules governing the interaction of bases leads to structural promiscuity, where a given sequence adopts several non-native folded conformations, or misfolds. Misfolding occurs most frequently at the level of secondary structure. In an effort to reduce the conformational heterogeneity commonly experienced by RNA, the modified nucleobase 8-bromoguanosine (8BrG) was introduced into oligonucleotides having the hairpin motif YNMG. Purine (=R) nucleotides with Br substituted for R H8 are known to preferentially adopt the syn conformation as nucleosides. The hairpin motif YNMG was chosen as a model system because it has a syn G at position four of the loop that is essential for thermodynamic stability. Thermodynamic and structural characterization of modified oligonucleotides with the tetraloop sequences UUCG, CGCG, and CGAG by UV thermal denaturation and NMR spectroscopy revealed that 8BrG substitution has a small effect upon the hairpin conformation, while the duplex conformation is strongly destabilized, thus inhibiting dimerization. These results support a model in which 8BrG-substituted YNMG sequences preferentially adopt the hairpin conformation, likely due to duplex destabilization. Substantial destabilization of UUCG with 8BrG incorporated into the helical hairpin stem provides further support for a model in which 8BrG substitution shifts the hairpin-duplex equilibrium towards the hairpin conformation by destabilizing the duplex. Catalytic RNA, or ribozymes, often experience conformational heterogeneity, which limits their functional efficacy. Substitution of 8BrG into ribozymes having syn purines in their active sites may limit misfolding, and enhance stability and catalytic rates. The lead-dependent ribozyme, or leadzyme, is an ideal system for testing the utility of 8BrG because solution, crystal, and theoretical structures of this ribozyme differ in the orientation of three G residues in the active site. A brief overview of current results is presented for 8BrG incorporation in the active site of the leadzyme. Modified nucleotides allow fundamental energetic and kinetic properties of nucleic acids to be probed. Incorporation of 8BrG for the syn G at loop position four of the YNMG RNA hairpin motif results in enhanced stability relative to the unmodified hairpin based on UV thermal denaturation. The lack of a discernable effect upon the hairpin conformation in NMR spectra suggests the enhanced stability of the modified sequence arises from a denatured state effect. Laser temperature-jump (T-jump) experiments support this notion, as unmodified and 8BrG-substituted UUCG have similar unfolding rates, but the folding rate of the modified tetraloop is enhanced approximately 3-fold. Based on 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. In addition to finding that 8BrG incorporation enhanced the stability and rate of folding, laser T-jump experiments revealed that UUCG hairpins closed by a CG base pair fold faster and unfold slower than hairpins closed by a GC base pair. Electrostatic isopotential contours calculated using solutions to the nonlinear Poisson-Boltzmann (NLPB) equation indicated that favorable arrangement of electrostatic potentials in the denatured state may drive faster folding for stable hairpins closed by a CG base pair. This finding suggests that favorable electrostatic interactions in the native and denatured states may enhance stability and folding. Electrostatic interactions are an important driving force in a variety of biological processes. There is increasing evidence that suggests electrostatics plays an important role in RNA stability, folding and catalysis. In particular, electrostatic interactions are likely to play a role in the modulation of pKa values in RNA, and are known to affect the binding of metal ions to RNA. In an effort to understand the mechanism of catalysis by the hepatitis delta virus ribozyme and to identify putative Mg2+ binding sites, surface electrostatic potential maps were calculated on the self-cleaved form of the ribozyme using the NLPB equation. These calculations revealed several patches of highly negative potential, one of which is present in a cleft near the putative general acid, N4 of C75. These calculations suggest that distinct catalytic and structural metal ion sites exist on the ribozyme, and that the negative potential at the active site may help shift the pKa for N3 of C75 toward neutrality.