All tRNA sequences natively fold into the well-known clover leaf structure. However most folding algorithms have severe problems predicting that structure. We will test several folding algorithms for their capabilities to predict the correct clover leaf shape. All of the next examples will use these two Human tRNAs.

1. Fold the two human tRNA sequences on the RNAfold Webserver. How good is the prediction? Can you recognize the clover leaf?
2. The programm RNAsubopt (at the GZI use /vol/biotools/bin/RNAsubopt within an Xterm) enumerates all suboptimal structures within a given energy range below the optimal structure. Try to find the clover leaf in the suboptimal folding space. Visualize the structures with RNAmovies (The correct format for RNAmovies is a fasta header in the first line, the plain RNA sequence as a second line and pure dot-bracket structures in the following lines; without energy values or other columns. Here is an example file.). If you can't see the cloverleaf structure, you must enumerate more suboptimal solutions. Call RNAsubopt with the parameter -e 5
3. Now, fold the sequences with RNAshapes in "Shape folding" mode. Look at which position the clover leaf shape occurs for each of the two sequences.

Hint: Don't dismiss your results, you will need them in the exercises for the structure comparision topic!