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1.
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Determine the predicted free energies of formation of the optimal secondary structures of different RNA's based on the
assigned dinucleotides.
First use the
mfold RNA scoring tables to make your own assessment as to what you think the free energies ought to be.
Then use
mfold
to get another opinion.
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1a.
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The RNA should consist of 10
repetitions of the dinucleotide, followed by 10 N's, followed by the inverse of the first 20
nucleotides. For example, considering the dinucleotide "AC", you would test:
ACACACACACACACACACACNNNNNNNNNNGTGTGTGTGTGTGTGTGTGT
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1b.
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The RNA
should consist of 10 repetitions of the first nucleotide followed by 10
repetitions of the second, followed by 10 N’s, followed
by the inverse of the first 20 nucleotides. For example, considering the
same dinucleotide “AC”, you would test:
AAAAAAAAAACCCCCCCCCCNNNNNNNNNNGGGGGGGGGGTTTTTTTTTT
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1c.
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Enter both free-energy values into tables that will represent findings from everyone. Use for this purpose the FILL-IN-ENERGY-TABLE function. What general conclusions do you draw from a comparison of the free energies of the different oligonucleotides?
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2.
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How can you tell whether a predicted structure is biologically significant?
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2a.
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Use
mfold
to predict the secondary structures of mystery sequences,
seq1.
Does it look significant to you? Note the free energy.
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2b.
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Predict the secondary structure of a random sequence with the same nucleotide composition as
seq1. How does it look? Note the free energy.
Possibly useful function:
SHUFFLE (in STRING/SEQUENCES, String-production)
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2c.
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Assign the free energy of the folding of the random sequence to a variable called energy. All of our values will be aggregated. What general conclusions do you draw from a comparison of the free energies of the random oligonucleotides to that of seq1?
Possibly useful function:
DEFINE (in DEFINITION)
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4.
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Is RNAz, implemented in BioBIKE as FIND-CONSERVED-RNA-IN, capable of (re)discovering
small noncoding RNAs in real genomes?
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4a.
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Pick out a test case. Choose your favorite gene amongst the noncoding genes of Prochlorococcus marinus MED4 (nicknamed PMED4).
Possibly useful functions:
NON-CODING-GENES-OF (in GENOME)
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4b.
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Create the gene anchors, between which you will search for a putative noncoding RNA. Define a variable, upstream-anchor, as the gene upstream from the test gene. Similarly, define a variable, downstream-anchor, as the gene downstream from the test gene. These will be used to find regions in other organisms where the putative RNA gene might lie.
Possibly useful functions:
GENE-UPSTREAM-OF
(in GENES/PROTEIN, Gene-neighborhood)
GENE-DOWNSTREAM-OF
(in GENES/PROTEIN, Gene-neighborhood)
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4c.
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Create the sequence between the anchor genes that you'll search for the putative noncoding RNA.
Possibly useful functions:
SEQUENCE-FROM
(in your FUNCTIONS menu)
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4d.
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Create a list of possible target sequences in several organisms (not to exceed 6). To do this, create a FOR-EACH loop, where you loop for each organism within a list of organisms. Within the loop (e.g. in the body of the loop), Define one variable as the ortholog of the upstream-anchor in the current organism, a second variable as the ortholog of the downstream-anchor in the current organism, and a third variable as the sequence from the first gene to the second. Within the Results section, collect this sequence. I suggest you use the first six organisms of *marine-prochlorococcus*.
Possibly useful functions:
FOR-EACH
(in FLOW/LOGIC)
*marine-prochlorococcus* (in DATA, Marine Unicellular Cyano)
FIRST (in LISTS-TABLES, List-extraction)
ORTHOLOG-OF (in GENES/PROTEIN)
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4e.
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Try to find the conserved RNA by running RNAZ (as implemented through FIND-CONSERVED-RNA-IN) over the list you created, or slices within the list. Note that FIND-CONSERVED-RNA-IN allows you to work with a FROM/TO range within the aligned sequences.
Possibly useful functions:
FIND-CONSERVED-RNA-IN (in STRING/SEQUENCES, Bioinformatic Tools)
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5.
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You have experimentally identified two sRNAs
(sRNA1 and
sRNA2)
in the cyanobacterium Prochlorococcus marinus MED4 (nicknamed PMed4). Do the RNAs exist in other related cyanobacteria: PMIT9313 and
S8102?
Possibly useful functions:
SEQUENCE-SIMILAR-TO (in GENES/PROTEIN)
CONTEXT-OF (in GENES/PROTEIN,
Gene-neighborhood)
SEQUENCE-UPSTREAM-OF (in GENES/PROTEIN,
Gene-neighborhood)
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