Thermodynamics of RNA-RNA binding

Background: Reliable prediction of RNA–RNA binding energies is crucial, e.g. for the understanding on RNAi, microRNA–mRNA binding and antisense interactions. The thermodynamics of such RNA–RNA interactions can be understood as the sum of two energy contributions: (1) the energy necessary to ‘open’ the binding site and (2) the energy gained from hybridization. Methods: We present an extension of the standard partition function approach to RNA secondary structures that computes the probabilities Pu[i, j] that a sequence interval [i, j] is unpaired. Results: Comparison with experimental data shows that Pu[i, j] can be applied as a significant determinant of local target site accessibility for RNA interference (RNAi). Furthermore, these quantities can be used to rigorously determine binding free energies of short oligomers to large mRNA targets. The resource consumption is comparable with a single partition function computation for the large target molecule. We can show that RNAi efficiency correlates well with the binding energies of siRNAs to their respective mRNA target

Translational Control by RNA-RNA Interaction: Improved Computation of RNA-RNA Binding Thermodynamics

The thermodynamics of RNA-RNA interaction consists of two components: the energy necessary to make a potential binding region accessible, i.e., unpaired, and the energy gained from the base pairing of the two interaction partners. We show here that both components can be efficiently computed using an improved variant of RNAup. The method is then applied to a set of bacterial small RNAs involved in translational control. In all cases of biologically active sRNA target interactions, the target sites predicted by RNAup is in perfect agreement with literature. In addition to prediction of target site location, RNAup can be also be used to determine the mode of sRNA action. Using information about target site location and the accessibility change resulting form sRNA binding we can discriminate between positive and negative regulators of translation

The Effect of RNA Secondary Structures on RNA-Ligand Binding and the Modifier RNA Mechanism: A Quantitative Model

RNA-ligand binding often depends crucially on the local RNA secondary structure at the binding site. We develop here a model that quantitatively predicts the effect of RNA secondary structure on effective RNA-ligand binding activities based on equilibrium thermodynamics and the explicit computations of partition functions for the RNA structures. A statistical test for the impact of a particular structural feature on the binding affinities follows directly from this approach. The formalism is extended to describing the effects of hybridizing small \modifier RNAs' to a target RNA molecule outside its ligand binding site. We illustrate the applicability of our approach by quantitatively describing the interaction of the mRNA stabilizing protein HuR with AU-rich elements [Meisner et al. (2004), Chem. Biochem. in press]. We discuss our model and recent experimental findings demonstrating the ffectivity of modifier RNAs in vitro in the context of the current research activities in the field of non-coding RNAs. We speculate that modifier RNAs might also exist in nature; if so, they present an additional regulatory layer for fine-tuning gene expression that could evolve rapidly, leaving no obvious traces in the genomic DNA sequences

Common physical basis of macromolecule-binding sites in proteins

Protein–DNA/RNA/protein interactions play critical roles in many biological functions. Previous studies have focused on the different features characterizing the different macromolecule-binding sites and approaches to detect these sites. However, no common unique signature of these sites had been reported. Thus, this work aims to provide a ‘common’ principle dictating the location of the different macromolecule-binding sites founded upon fundamental principles of binding thermodynamics. To achieve this aim, a comprehensive set of structurally nonhomologous DNA-, RNA-, obligate protein- and nonobligate protein-binding proteins, both free and bound to their respective macromolecules, was created and a novel strategy for detecting clusters of residues with electrostatic or steric strain given the protein structure was developed. The results show that regardless of the macromolecule type, the binding strength and conformational changes upon binding, macromolecule-binding sites are energetically less stable than nonmacromolecule-binding sites. They also reveal new energetic features distinguishing DNA- from RNA-binding sites and obligate protein- from nonobligate protein-binding sites in both free/bound protein structures

The U1A/U2B /SNF Family of RNA Binding Proteins: Evolution of RNA Binding Specificity and Contributions of Heterotropic Linkage to snRNP Protein Partitioning

The U1A/U2B /SNF is a family of RNA binding proteins that is a highly conserved throughout eukaryotes. These proteins are found in the U1 and/or U2 splicing snRNPs (small nuclear ribonucleoprotein particles). In humans, U1A and U2B specifically bind to the U1 and U2 snRNAs, respectively. The Drosophila genome codes for SNF, an essential protein that localizes to both the U1 and U2 snRNP. While a specific splicing functions for these proteins have not been determined, their conserved snRNP localization suggests an important splicing-related function. The difference in protein number and partitioning between Drosophila and humans suggested that these proteins may use different RNA binding mechanisms to function in their cellular contexts. This work begins by exploring some of the differences amongst human U1A, U2B , and Drosophila SNF. The thermodynamics of the RNA-protein interactions also reveal substantial differences in the RNA binding mechanisms of these proteins. Further studies investigate the evolution of this protein family in metazoans. Reconstructing the protein phylogeny permitted resurrection of ancestral proteins. This led to the discovery that the last common ancestor of humans and Drosophila had a single U1A/U2B /SNF family homolog. This protein had RNA binding properties that most closely resemble those of Drosophila SNF. Evolution of protein motions and RNA binding specificity toward the defining characteristics of modern vertebrate proteins is also examined. Finally, linkage effects between protein-protein and protein-RNA interactions are analyzed. U2A\u27; is a U2 snRNP-specific protein that binds to U2B ; in humans and SNF in Drosophila. In Drosophila, large, positive linkage was only seen between U2A\u27-SNF and SNF-U2 snRNA binding. The RNA dependence of enhancement for SNF binding to U2A\u27 can explain the observed protein partitioning of U2A\u27 in vivo. For the more complicated human system, which contains two SNF homologs, substantial contributions to protein partitioning come from differences in both intrinsic RNA-protein binding affinities and differences in protein-U2A\u27 binding affinities. RNA dependence of the linkage parameter also contributes to protein partitioning. The binding parameters can explain U2A\u27 protein partitioning, and the presence of U2A\u27 reinforces U1A and U2B partitioning to their respective snRNAs. These linkage studies have important implications for the assembly of RiboNucleoprotein Particles, macromolecular complexes that are fundamental to many cellular activities

Nucleic Acid Binding Thermodynamics and Functions of DNA Polymerases

This study examines several different linkages between the nucleic acid binding thermodynamics and the functions of three different DNA polymerases. The focuses are correlation of the DNA binding thermodynamics and the functional behavior of Klenow and Klentaq polymerases (from Escherichia coli and Thermus aquaticus, respectively), identification of factors that influence the proofreading activity of Klenow, and examination of HIV reverse transcriptase (HIV-RT) binding to different primer/template nucleic acid constructs. A comparison of the DNA binding thermodynamics and the incorporation activity of Klenow and Klentaq reveals that the enthalpic versus entropic balance upon binding may function as a modulator of the temperature dependence of the enzymatic activity. Both polymerases bind DNA with nanomolar affinity at significantly low temperatures, but have negligible enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding crosses zero and becomes favorable (negative). Proofreading activity improves the fidelity of DNA synthesis. Proofreading requires unwinding of the primer strand and shuttling of the 3’ terminus of the primer from the polymerization site to the proofreading site. The binding of Klenow to matched and mismatched primed-template DNA was examined by monitoring the steady state fluorescence intensity change of a 2-aminopurine base site-specifically substituted in DNA and reveals that both the equilibrium partitioning and the dynamic partitioning between sites are dependent on the absence, presence, and identity of specific divalent cations, as well as on the presence of mismatched bases at the primer/template junction. HIV-RT performs both DNA and RNA template directed DNA synthesis. Direct binding equilibria have been characterized for the interaction of HIV-RT with several different primer/template nucleic acid constructs across a range of KCl concentrations. The thermodynamic affinities of the two homoduplexes (DNA/DNA and RNA/RNA) are shown to be nearly identical, while binding of the heteroduplexes is significantly tighter. At least two different modes of nucleic acid binding are revealed by the thermodynamic salt linkages of binding, and these different thermodynamic binding modes correlate with different recently structurally elucidated binding modes

Molecular principles underlying dual RNA specificity in the Drosophila SNF protein

The first RNA recognition motif of the Drosophila SNF protein is an example of an RNA binding protein with multi-specificity. It binds different RNA hairpin loops in spliceosomal U1 or U2 small nuclear RNAs, and only in the latter case requires the auxiliary U2A′ protein. Here we investigate its functions by crystal structures of SNF alone and bound to U1 stem-loop II, U2A′ or U2 stem-loop IV and U2A′, SNF dynamics from NMR spectroscopy, and structure-guided mutagenesis in binding studies. We find that different loop-closing base pairs and a nucleotide exchange at the tips of the loops contribute to differential SNF affinity for the RNAs. U2A′ immobilizes SNF and RNA residues to restore U2 stem-loop IV binding affinity, while U1 stem-loop II binding does not require such adjustments. Our findings show how U2A′ can modulate RNA specificity of SNF without changing SNF conformation or relying on direct RNA contacts