, 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 an assignment in a lecture course. The websites listed above are free to use and have access to the latest entries in the nucleic acid sequence data bank, but the following websites can also be used as alternates if deemed necessary:
http://embnet.vital-it.ch/software/ClustalW.html (CLUSALW to perform sequence alignments)
http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi (Vienna Package to predict nucleic acid secondary structure)
Nucleic acids review
- Nucleic acids are polymers of_____________________________. ( / 1 pt.)
- Nucleic acid polymers (strands) go from ___ to 3' direction. ( / 1pt.)
- GC pairs have _________H-bonds while AT pairs have _______H-bonds, therefore AT pairs are ___________ (choose more or less) stable than GC pairs. ( / 3 pts.)
- Draw the structure of ATP. ( / 1pt.)
- Indicate sugar, phosphate and the nucleobase on the nucleotide below. ( /3 pts.)
- Write the complementary sequence for the following nucleotide. ( / 1 pt.)
5’ GCCGATA 3’
Questions regarding “Identifying invariable blocks in the toxin sensor”
- Circle “invariable blocks” in the following sequence alignment. ( /2pts.)
- Circle conserved structural elements (structures that all of these RNAs have in common). ( / 2pts.)
Identifying Invariable Blocks in the Toxin Sensor
Lab Report Outline and Point Distribution
- Define the goal of the experiment (3 pts.).
- Copy of multiple sequence alignment (3 pts.).
- Circle each invariable block (3 pts.).
- Secondary structure predictions for Bacillus subtilis, Staphlococcus saprophyticus and Gloebacter violaceus ykkCD sensor RNAs. (3 pts. each = 9 pts. total).
- Circle the conserved structural elements for the three structural predictions performed (3 pts.).
- Show structural prediction for mutant 1 (2 pts.).
- Comment on why you believe that your mutation caused these specific structural changes (5 pts.).
- Show the structural prediction for mutant 2 and mutant 3 (4 pts.).
- Comment on why you believe that mutant 3 restored the original structure (5 pts.).
- Based on your experience with mu