Exercise 6

Key - Exercise 6 - BIMM 141 - Spring. 2001

Part A: 5 pts
Part B: 55 pts
Part C: 5 pts
Part D: 25 pts
Part E: 85 pts

Total Points: 175 pts

{A. Locate an RNA sequence of interest .}

{Enter a description of your molecule and the known base-pairing into your notebook.}
[5 pts]

{B. MFOLD - multiple sub-optimal RNA structures.}

{1. Learn about the GCG programs MFOLD in GENHELP.}

{2. Use PLOTFOLD to examine the squiggles, mountains, circles, domes, and dotplot output from MFOLD.} [25 pts]

{3. Use PLOTFOLD to examine at least five of the sub-optimal folds produced by MFOLD. What graphics output is most useful in this comparison?  Which one of these structures, if any, corresponds to the known structure of your RNA? } [30 pts]

{C. MFOLD parameters: constraining the folding pattern.}

{1. Based on your knowledge of the biology of your molecule (and/or the annotation in the sequence file), use some of the parameters of MFOLD: /REMOVE /PREVENT /FORCE.} [5 pts]

{D. Using the GCG programs STEMLOOP and DOTPLOT for RNA structures.}

{1. Learn about the GCG STEMLOOP program using GENHELP.  Analyse your sequence using STEMLOOP and display your results using DOTPLOT.} [5 pts]

{2. Compare these results with those from MFOLD as displayed using PLOTFOLD.} [10 pts]

{3. Attempt to find a set of parameters for STEMLOOP which yields output in which you can see the stems predicted using MFOLD.} [10 pts]

{E. Questions.}

1. What are the problems with the SQUIGGLES representation for RNA structures? [5 pts]

SQUIGGLES can be misleading about whether two structures are the same or not depending on the angle and positions of the drawn stem-loop structures. That is, two structures which look dissimilar may be mathematically equivalent if rotated or the stemloops bent at another angle.

 

2. Name a representation that corrects these problems? [5 pts]

Any of the other depictions discussed in class, MOUNTAINS, DOMES, CIRCLES correct this problem.

 

3. What is the difference between an internal loop and a bulge loop? [5 pts]

An internal loop comprises bases on both strands, while a bulge loops has unpaired bases in only one strand.

 

4. What are the advantages of MFOLD relative to STEMLOOP? [15 pts]

1. MFOLD uses Zuker's energy rules to determine folding structures while STEMLOOP looks only for potential stem-loop structures based on whether a sequence can fold back on itself using very simple base pairing energetics.
2. STEMLOOP doesn't give any indication of the relative stability/energy level of a given stem structure while MFOLD does calculate the stability/energy.
3. STEMLOOP treats each stem-loop as an independent structure and shows all possible stem-loop structures while MFOLD evaluates each structure vs. structures in the rest of the RNA.
4. MFOLD considers bulges and internal loops while STEMLOOP does not.
5.MFOLD can constrain the base pairing (/FORCE, /REMOVE, /PREVENT), while only the window/stringency of stems can be specified in STEMLOOP.
6. MFOLD produces a more diverse set of folding representations.

5. What are the disadvantages of MFOLD relative to STEMLOOP? [5 pts]

 

6. What kinds of structures cannot be found by folding programs such as MFOLD? [5 pts]

Knots and pseudo knots can not be evaluated by MFOLD. MFOLD strictly deals with secondary structures; knots and pseudoknots are defined as tertiary structures.

 

7. In general, which will destabilize a structure more: an internal loop or a bulge loop. Explain your

answer and include energies. [5 pts]

Internal/bulge loop energies are always destablizing (positive free energy). Depending on the number of unpaired bases, the interior base pair, and the exterior base pair. Internal loops are generally more unstable since some small bulge loops may have stacking energies (stablizing) added.

 

8. What are the main energy terms considered in computer prediction of RNA folding? [10 pts]

The main energy terms for RNA folding are:
(1) hydrogen bonding of base pairs (negative/stabilizing energy);
(2) stacking energy of neighboring base pairs (generally negative/stabilizing energy);
(3) loops (generally positive/destablizing energy). Loops may be bulge, interior or multiloops.
These terms can be summarized in the following equation:
DG = DGstack + DGbulge + DGhairpin + DGinternal loop + DGmultibranch
where DGstack is the energy (negative/stabilizing) of stacked basepairs and the rest of the terms are positive/destablizing.

9. List the favorable energy terms (those that stabilize structures) in RNA folding? [5 pts]

The stacking energy is the only favorable term.

 

10. When is a GU basepair less stable that of a AU basepair? [10 pts]

GU basepairs adjacent to AU or UA basepairs are less stable (-1.2 kcal/mol) than AU basepairs adjacent to GC or CG basepairs (-2.5 and -2.0 kcal/mol) at 20 C (handout notes). At 37 C, the differences are greater, however the same pattern is seen, with both AU and GU basepairs being more stable when adjacent to GC or CG basepairs (-1.2 to -2.3 kcal/mol) than when adjacent to AU, UA, GU or UG basepairs (-0.5 to -0.9 kcal/mol) as shown in the lecture notes.

 

11. What laboratory methods might one use to test the correctness of a predicted structure? [5 pts]

Nuclease digestion and crosslinking studies are laboratory experiments that can identify some of the bases that are paired and unpaired. Ultimately x-ray crystallography and NMR spectroscopy can determine the three-dimensional structure but are very difficult with RNA.

 

12. What non-laboratory (e.g., computational) methods might on use to test the correctness of a predicted structure? [5 pts]

The primary non-laboratory methods for examining structure is prediction by homology to other known structures based on the co-variation of paired residues, and conservation of base-paired structures across large phylogenetic distances.

 

13. What is a P-num plot and why would one use it? [5 pts]