 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 mutants 1 and 2 explain why changing structure and sequence at the same time makes it hard to interpret the functional effect of a mutation (aka what was the reason for designing mutant 3) (7 pts.).
- Choose a mutant (from the three designed here) that you would like to test experimentally. Provide a brief explanation for why you chose this mutation (6 pts.).
BLAst Problem Set
Define or describe the following terms as used in BLAST:
- E (Expect or Expectation value)
- Max Score
Find and report the following:
- The number of identities in the alignment between the query and the subject, Cyanothece sp. PCC7425
- The percent of coverage between the query and the subject, Sulfuricurvum kujiense DSM16944
- The number of gaps in the alignment between the query and the subject, Clostridium kluvyeri DSM555
- The Expect value in the alignment between the query and the subject, Exiguobacterium sibiricum 255-15, complete genome.
- The percent of maximum identity between the query and the subject, Geobacillus sp Y412MC52
Find and interpret the following:
- Find the E value for the alignment between the query and the subject, Bacillus subtilis subsp. spizizenii str. W23, complete genome. How do you specifically interpret this number? (What does this number mean?)
- Find the E value for the alignment between the query and the subject, Pseudomonas stutzeri DSM 4166. How do you specifically interpret this number? (What does this number mean?)
- For the alignment between the query and the subject, Paenibacillus sp. JDR-2, complete genome, there are fourteen gaps reported yet only five gap openings are shown in the alignment. Is this a mistake? Briefly explain.
General BLAST interpretation:
- The search using megablast gave far fewer matches than the search using blastn. Check the Algorithm Parameters for both searches. Could the difference in word length explain the difference in matches? Briefly explain.
- The alignment with the subject, Clostridium cellulovorans 743B complete genome, has a smaller % identity than the subject, Lys