pset3_ans - MIT Department of Biology 7.28, Spring 2005 -...

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MIT Department of Biology 7.28, Spring 2005 - Molecular Biology Question 1. You are interested in understanding the residues of a specific group I intron that function specifically in catalysis of the splicing reaction. To probe this question, you mutagenize the intron and, using an in vivo screen, isolate mutant variants that are splicing-defective. Because group I introns contain many regions of secondary structure that are important for folding of the RNA but probably are not directly involved in catalysis, you decide to initially examine your mutant RNAs for those defective in folding. These mutants will be set aside, as they are unlikely to have specific defects in the catalytic steps. 1a You find that it is relatively easy to isolate the unprocessed RNA from your mutant cell lines. With this in mind, suggest a type of analysis you could use to probe for mutants that have defects in RNA folding. Explain what controls you will use, and how you will decide if the mutant is a folding defect. You can identify mutants in RNA folding by isolating the unprocessed RNA from wt and each of your mutants (1 pt), end-labeling it, digesting each with either S1 nuclease or snake venom RNAse individually (1 pt), and running the products out on a denaturing gel (1 pt). S1 nuclease will digest ssRNA and SV will digest dsRNA (1 pt). You will compare the banding pattern in each of the mutants to the wild type pattern. Any ‘footprints’ that differ from the wt will indicate a mutant in RNA folding (2 pts). 1b After setting aside the folding mutants, you wish to further screen your mutants for those that are good candidates for having specific catalytic defects. You decide it will be easier to look for mutants that have defects in the second splicing step. Your idea is to look for mutant RNAs that accumulate the intermediates expected of an RNA that can catalyze the first step successfully, but show a complete defect in the second step. Given the schematic of the full length pre-RNA shown below, draw the structure of the RNAs you would expect to see after an in vitro splicing reaction for a mutant RNA that is defective in the second step. G-O-P-O----------------Exon2-----3’ (3 pts) 5’----Exon1----3’OH AND 1
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Also, in addition to your purified pre-mRNA, what components would you add to the in vitro splicing reaction? 1. (2 pts) Guanine nucleoside or nucleotide 2. (1 pts) Divalent cations (e.g. Mg ++ or Mn ++ ) 3. (1 pts) Monovalent cations (e.g. Na + or K + ) 1c Using the strategy outlined above, you successfully identify several mutant RNAs that are specifically defective in carrying out the second splicing reaction. You are most interested in mutants that identify residues that are truly part of the active site that catalyzes this reaction. Therefore, you next want to determine if the mutant RNAs are or are not defective in the tertiary structure rearrangement that is necessary prior to the second splicing step, as mutants defective in this rearrangement reaction are less
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pset3_ans - MIT Department of Biology 7.28, Spring 2005 -...

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