Ab initio RNA folding

RNA molecules are essential cellular machines performing a wide variety of functions for which a specific three-dimensional structure is required. Over the last several years, experimental determination of RNA structures through X-ray crystallography and NMR seems to have reached a plateau in the number of structures resolved each year, but as more and more RNA sequences are being discovered, need for structure prediction tools to complement experimental data is strong. Theoretical approaches to RNA folding have been developed since the late nineties when the first algorithms for secondary structure prediction appeared. Over the last 10 years a number of prediction methods for 3D structures have been developed, first based on bioinformatics and data-mining, and more recently based on a coarse-grained physical representation of the systems. In this review we are going to present the challenges of RNA structure prediction and the main ideas behind bioinformatic approaches and physics-based approaches. We will focus on the description of the more recent physics-based phenomenological models and on how they are built to include the specificity of the interactions of RNA bases, whose role is critical in folding. Through examples from different models, we will point out the strengths of physics-based approaches, which are able not only to predict equilibrium structures, but also to investigate dynamical and thermodynamical behavior, and the open challenges to include more key interactions ruling RNA folding.Comment: 28 pages, 18 figure

Pathways and kinetic barriers in mechanical unfolding and refolding of RNA and proteins

Using self-organized polymer models, we predict mechanical unfolding and refolding pathways of ribo-zymes, and the green fluorescent protein. In agreement with experiments, there are between six and eight unfolding transitions in the Tetrahymena ribozyme. Depending on the loading rate, the number of rips in the force-ramp unfolding of the Azoarcus ribozymes is between two and four. Force-quench refolding of the P4-P6 subdomain of the Tetrahymena ribozyme occurs through a compact intermediate. Subsequent formation of tertiary contacts between helices P5b-P6a and P5a/P5c-P4 leads to the native state. The force-quench refolding pathways agree with ensemble experiments. In the dominant unfolding route, the N-terminal a helix of GFP unravels first, followed by disruption of the N terminus b strand. There is a third intermediate that involves disruption of three other strands. In accord with experiments, the force-quench refolding pathway of GFP is hierarchic, with the rate-limiting step being the closure of the barrel.Comment: 33 pages 7 figure

Single molecule studies on the dynamics of the transcription initiation complex of yeast mitochondria

Department of Biomedical EngineeringThe transcription initiation complex in the yeast mitochondria of Saccharomyces cerevisiae comprises the RNA polymerase, Rpo41, the initiation factor, Mtf1, and the DNA including 6 base pair promoter sequence. The Mtf1 is known to recognize and help to open the promoter region during the initiation stage, but its exact role and mechanism still remains unclear. We designed a multi-color single molecule FRET assay to directly measure the dynamics of the complex during transcription initiation. The labels on the DNA report on its opening-closing dynamics, while the label on Mtf1 report on the recruitment, dynamics, and dissociation of the initiation factor. From these measurements, we can correlate the promoter opening dynamics, factor binding/dissociation, and the transition to the elongation phase. Mtf1 is also associated with controlling the production of abortive RNA transcripts. We observed the scrunching motion during transcription by stepping along the DNA template with various combinations of nucleotide substrates. The FRET distribution shifted toward the high FRET region as we stepped further. From these observations, we propose a mechanistic model of the transcription initiation in the yeast mitochondria.ope