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RNA DBP: modeling and dynamics of RNA Russ Altman Vijay Pande Outline Introduction to RNA Introduction to tetrahymena intron Challenges in DBP Simulation of RNA Discussion Central Dogma of Biology QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Growing protein chain RNA Function DNA RNA polymerase Messenger RNA 20 Transfer RNA (tRNA) charged with amino acids Messenger RNA Ribosome (rRNA) 0. Like DNA, RNA contains 4 subunits (AUGC). It is less stable than DNA, so is not a storage media. 1. the DNA code a gene is copied into messenger RNA (mRNA) 2. mRNA is the version of the genetic code translated at the ribosome. 3. the ribosome is made up RNA (ribosomal RNA or rRNA) 4. The individual amino acids are brought to the ribosome, as it reads the mRNA, by molecules called transfer RNAs (tRNA) RNA has 3D structure Primary Structure AUUCGGCGACGAAU AUUCG G G UAAGC A C Secondary Structure Tertiary Structure RNA folding is usually nested 1 76 Growing protein chain RNA Function DNA RNA polymerase Messenger RNA 20 Transfer RNA (tRNA) charged with amino acids Messenger RNA Ribosome (rRNA) RNA polymerase QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Growing protein chain RNA Function DNA RNA polymerase Messenger RNA 20 Transfer RNA (tRNA) charged with amino acids Messenger RNA Ribosome (rRNA) http://www.wadsworth.org/BMS/ http://www-smi.stanford.edu/projects/helix/ribo.html QuickTime™ and a QuickTime™ and a TIFF (Uncompressed) decompressor TIFF (Uncompressed) decompressor are needed see this to picture. aretoneeded see this picture. Genetic Code (T=U here) (e.g. Tyrosine = UAU or UAC) Growing protein chain RNA Function DNA RNA polymerase Messenger RNA 20 Transfer RNA (tRNA) charged with amino acids Messenger RNA Ribosome (rRNA) mRNA (yellow) interacting with A and P site tRNA anticodons Growing protein chain RNA Function DNA RNA polymerase Messenger RNA 20 Transfer RNA (tRNA) charged with amino acids Messenger RNA Ribosome (rRNA) Biological significance of RNA folding? mRNA takes on 3D structure as it is produced (cotranscriptional folding), and this may affect: stability within cell speed of translation frequency of translation interactions with other molecules (regulation of other mRNA, e.g.) The possibility that an RNA takes on a 3D structure that is recognized or used by the cell constantly vexes biologists who see unexpected phenomena. Tetrahymena Intron Dan Herschlag, Biochemistry PI Biological Aims 1. Determine mechanism of rapid electrostatic collapse, structure of collapsed particle, physical forces 2. Determine spectrum of intermediates that are formed, and dynamic properties. 3. Determine landscape for folding, and features critical for driving down native pathways. Experimental Modalities Hydroxyl radical footprinting (equilibrium and time-resolved) Small angle x-ray scattering (equilibrium and time-resolved) Single molecule fluoresence energy transfer (FRET) Single molecule force measurements Biochemical modifications to perturb structure and folding (introduce point mutations or entire extra pieces) Coarse Grained L-21 and P4P6 Gnomad L-21, 409 atoms RMSD 1.2Å 132 secs CPU (Xportal1) P4P6, 409 atoms RMSD 15Å 45 secs CPU (Xportal1) Hydroxyl Radical Footprinting + •OH Lot’s of smaller RNA molecules 32P Run on a Gel The more surface exposed nucleotides will be more reactive A170 U169 G168 G167 A166 U165 A164 C163 A162 A161 G160 C159 U158 A157 C156 G155 U154 U153 C152 G151 C150 C149 C148 A147 C146 G145 C144 Site specific kinetics k1 k2 k1 k2 U I1 F Small angle x-ray scattering (SAXS) Single molecule measurements Biochemical modifications Computational Challenges Moving to mesoscale for computability Optimization of models to be consistent with data Constrained dynamics (drive trajectory through milestone structures) Data visualization and ensemble mapping (data is aggregate, models are single)