1. You have discovered a novel sigma factor in coli cells called ?25 Based on da
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Question
1. You have discovered a novel sigma factor in coli cells called ?25 Based on data from a DNase foot-printing assay you have determined that ?25 binds to consensus sequences at positions 15 and -30 upstream of genes involved in the stress response to lambda bacteriophage infections, including gene z. You hypothesize that in order for gene z to be transcribed the ?25 protein must bind to the -15 and -30 regions. What approach could you use to test this hypothesis? You have already cloned the promoter region (containing the -15 and -30 regions) and the coding region for gene z into pUC19. What molecular techniques could you use to test your hypothesis? What results would you need to get to support your hypothesis? (4 points) dependent termination (2 points). 3. Given the wobble base pairing, only 9 tRNAs are needed to encode the 4 amino acids Ser, Pro, Trp, and Leu. List the mRNA codons for the 4 amino acids and necessary 9 tRNA anti-codons. (4 points) 4. What is the amino acid sequence of the peptide encoded by the following mRNA sequence? Which positions in the mRNA can tolerate a mutation without resulting in a change in amino acid sequence? What changes are tolerated at each of these positions? (2 points) 5-AUGACCAAAUGGGCG-3'Explanation / Answer
This hypothesis can be solved by two method.
We know the DNA sequences of promotor and gene z. From the DNA footprinting assay it is confirm that sigma factor binds to the promotor.
Hypothesis is Binding of Sigma factor to the promotor sequence is essential for gene z transcription.
Experiment : Mutate the promotor sequence at -15 and -30 upstream of the gene.
Reason: When the promotor is mutated, the DNA binding domain of sigma factor cant bind with the promotor.
Expected result: If transcription of gene z occur it means that non-binding of sigma factor to promotor sequence does not affect gene z transcription.
If transcription of gene z doesnt occur it means that non- binding of sigma factor to promotor sequence plays a vital role in gene z transcription.
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This hypothesis can also be tested by altering the DNA binding motif of the sigma factor protein which in turn leads to non-binding of sigma factor to the wild-type promotor sequence.
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