2. RNA DBP: modeling and dynamics of RNA Russ Altman Vijay Pande

3. Outline  Introduction to RNA  Introduction to tetrahymena intron  Challenges in DBP  Simulation of RNA  Discussion

4. Central Dogma of Biology

5. RNA Function Growing protein chain 20 Transfer RNA (tRNA) charged with amino acids Ribosome (rRNA) DNA Messenger RNA RNA polymerase Messenger RNA

6.  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)

7. RNA has 3D structure AUUCGGCGACGAAU G AUUCG UAAGC C G A Primary Structure Secondary Structure

8. Tertiary Structure

10. RNA folding is usually nested 1 76

11. RNA Function Growing protein chain 20 Transfer RNA (tRNA) charged with amino acids Ribosome (rRNA) DNA Messenger RNA RNA polymerase Messenger RNA

12. RNA polymerase

13. RNA Function Growing protein chain 20 Transfer RNA (tRNA) charged with amino acids Ribosome (rRNA) DNA Messenger RNA RNA polymerase Messenger RNA

14. http://www.wadsworth.org/BMS/ http://www-smi.stanford.edu/projects/helix/ribo.html

16. Genetic Code (T=U here) (e.g. Tyrosine = UAU or UAC)

17. RNA Function Growing protein chain 20 Transfer RNA (tRNA) charged with amino acids Ribosome (rRNA) DNA Messenger RNA RNA polymerase Messenger RNA

21. mRNA (yellow) interacting with A and P site tRNA anticodons

22. RNA Function Growing protein chain 20 Transfer RNA (tRNA) charged with amino acids Ribosome (rRNA) DNA Messenger RNA RNA polymerase Messenger RNA

23. 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.

24. Tetrahymena Intron Dan Herschlag, Biochemistry PI

30. 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.

31. 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)

32. 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)

33. Hydroxyl Radical Footprinting + OH Lot’s of smaller RNA molecules 32 P Run on a Gel The more surface exposed nucleotides will be more reactive C144 G145 C146 A147 C148 C149 C150 G151 C152 U153 U154 G155 C156 A157 U158 C159 G160 A161 A162 C163 A164 U165 A166 G167 G168 U169 A170

34. Site specific kinetics

35. Small angle x-ray scattering (SAXS)

36. Single molecule measurements

37. Biochemical modifications

38. 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)