le block of your choosing, or modify its sequence.
2.7 Identifying Conserved Structural Elements
Besides invariable blocks, conserved structural elements may also serve as hot spots for recognition. These are regions in the molecule that may have different sequence, but form the same structure. For example, both the GGGG AAAA CCCC and the AAAA GGGG TTTT nucleic acids form a hairpin even though they have very different sequence. To identify conserved structural elements in the ykkCD toxin sensor first you will predict the secondary structure of the ykkCD sensor RNA using ykkCD sequences from different organisms. Then you will select structural elements that are present in each organism based on visual inspection of the predicted structures. To test if a conserved structural element is important for toxin recognition, you will design a mutant that significantly alters a conserved element, then design another mutant that restores the original structure (compensatory mutation). If the structural element was important for toxin recognition then significantly changing the structure would destroy recognition, but restoring the original structure, albeit with altered sequence, will restore the ability of the sensor to recognize its target toxin.
Identifying invariable blocks
- Go to http://blast.ncbi.nlm.nih.gov/Blast.cgi.
- Click nucleotide blast.
- Paste the sequence below into the “Enter Query Sequence” window.
T G TA A AG T T T T C TAG G G T T C C G C AT G T C A AT T G AC AT G G AC
T G G T C C G AG AG A A A AC A C ATAC G C G TA A ATAG A AG C G C G T
- Choose “others” under “Choose Search Set/Database” and “Somewhat similar
sequences” under “Program Selection”.
- Hit BLAST.
You will see many sequences color-coded by the degree of similarity to the B. subtilis ykkCD RNA.
Find ykkCD sequences in the organisms below and paste them into your lab notebook!
Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus halodurans, Bacillus pumilus, Alkaliphilus oremlandii, Staphylococcus saprophyticus subsp, Symbiobacterium thermophilum
- Perform multiple sequence alignment on the selected sequences by going to
- Each sequence should be entered in a different row with designation preceding the sequence as follows:
>Sequence name (for example B.subtilis)
sequence (for example Tgtaaagt……)
- Hit “Start MultAlin”.
- Nucleotides colored in red represent invariable groups.
Identifying conserved structural element
- Go to http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form.
- Paste the RNA sequences below into the program window to predict their structure.
B subtilis sequence
Staphylococcus saprophyticus sequence
- Choose “Fold RNA”.
- To view predicted structures go to “View Individual Structures”. Select jpeg
Mutant design (design three mutants)
- Propose a mutant that changes a conserved sequence element of the toxin sensor (mutant 1).
- Predict the structure of this mutant using Mfold.
- If the mutant changed the structure of the sensor significantly (predicted structure is completely different from the original one) choose a different mutant until you find one that changed a conserved sequence without significantly changing the overall structure.
- Design mutants to test if a conserved structural element is important for toxin recognition by following the outline below.
- – Propose a mutant that significantly alters or eliminates a conserved structural element (mutant 2).
- – Design a compensatory mutant to restore the original structure (mutant 3).
Notes to the instructor
The experiment in Chapter 2 is designed to identify sequence conservation in the B subtilis tetracycline sensor RNA ykkCD. The same protocol with minimal modifications could be used to identify sequence conservation in any nucleic acid (regulatory RNA, promoter or ribozyme). Two students per computer works well to maximize peer interaction while still making sure that each student has a chance to intellectually contribute to the assignments. Tablets or smart phones may also be used to complete each task. This means, this experiment may be used as