151. Fluorescence Competition and Optical Melting Measurements of RNA Three-Way Multibranch Loops Provide a Revised Model for Thermodynamic Parameters†
- Author
-
Douglas H. Turner, David H. Mathews, Joshua M. Diamond, and Biao Liu
- Subjects
Base pair ,RNA Stability ,Molecular Sequence Data ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,Fluorescence ,Nucleic acid secondary structure ,03 medical and health sciences ,chemistry.chemical_compound ,Nucleic acid thermodynamics ,Transition Temperature ,Protein secondary structure ,030304 developmental biology ,0303 health sciences ,biology ,Base Sequence ,Ribozyme ,RNA ,0104 chemical sciences ,Crystallography ,chemistry ,Transfer RNA ,biology.protein ,Nucleic Acid Conformation ,Thermodynamics ,DNA - Abstract
RNA multibranch loops (junctions) are ubiquitous in known RNA secondary structures, including tRNA (1), rRNA (2,3), the HIV-1 genome (4), and ribozymes (5). They can be characterized by the number of helices forming the loop, the number of unpaired nucleotides located in the junction, the base pairs closing the junction, and other factors. Despite the prevalence, the factors that determine the stabilities of RNA multibranch loops are poorly understood. Research on DNA multibranch loops sheds some light onto factors that are likely to be important for RNA multibranch loops. The dependence of structure on the number and type of unpaired nucleotides in DNA multibranch loops has been studied extensively for loops of three, four, and five helices (6−11). DNA multibranch loops with unpaired purines in the junction have different stacking conformations than similar loops containing unpaired pyrimidines (6,7). Furthermore, the effects of unpaired nucleotides on the stability of the junction are dependent on the number of helices forming the junction (8). The presence of two unpaired nucleotides allows formation of a stable DNA three-way junction in which two of the helices coaxially stack, but unpaired nucleotides are not required to stabilize a four-way DNA junction (8−11). Approaches to estimate the free energy change of forming an RNA multibranch loop include (1) optimizing the prediction of known RNA secondary structures (12), (2) fitting experimental results (13−15), and (3) using a combination of these approaches (16). Knowledge-based statistical methods (17−19) could also be used. Coaxial stacking of helixes is observed in RNA multibranch loops (1,20−29) and can often be predicted on the basis of thermodynamics (30). RNA secondary structure prediction algorithms, such as MFOLD (12) and RNAstructure (15), use a simple model to predict multibranch loop stability (12,15). A more thorough understanding of factors contributing to RNA multibranch loop stability should improve the model used to predict stability and therefore the ability to accurately predict RNA secondary structure from sequence. The number of known RNA sequences is rapidly increasing, and it is important to understand structure−function relationships for RNA. Accurate prediction of secondary structure will accelerate discovery of these relationships and may help in assigning functions to RNA motifs and to new RNAs. This study uses a two-strand system modified from previous experiments (14) to measure by optical melting (31) and fluorescence competition assays (FCA) the influence of the number and the type of unpaired nucleotides on the stability of RNA three-way multibranch loops. A common motif in rRNAs, the loop E motif (3), was included in the investigation. Competition binding experiments with gel electrophoresis were used to determine the relative stabilities of DNA multibranch loops (8,32). FCA has been used to study nucleic acid thermodynamics, including stabilities of DNA duplexes (33) and of RNA pseudoknots (34).
- Published
- 2010