Exam 10: Transcription in Bacteria

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To demonstrate the self-cleaving activity and metabolite specificity of the novel riboswitch, we can design an experiment as follows:

1. In vitro transcription: First, we will synthesize the riboswitch sequence using in vitro transcription. This will allow us to obtain a pure sample of the riboswitch for our experiments.

2. Self-cleaving assay: Next, we will incubate the synthesized riboswitch with a buffer solution under different conditions. We will test for self-cleaving activity by adding a substrate that can be cleaved by the riboswitch. We will monitor the cleavage reaction using gel electrophoresis to visualize the cleaved products.

3. Metabolite specificity assay: To test for metabolite specificity, we will repeat the self-cleaving assay in the presence of different metabolites that the riboswitch is predicted to respond to. We will monitor the cleavage reaction in the presence of each metabolite and compare it to the reaction in the absence of any metabolite.

Sample positive results:
- In the self-cleaving assay, we observe a clear band corresponding to the cleaved product on the gel, indicating that the riboswitch is indeed capable of self-cleaving activity.
- In the metabolite specificity assay, we observe a significant increase in self-cleaving activity in the presence of a specific metabolite, while other metabolites have little to no effect on the cleavage reaction. This demonstrates the metabolite specificity of the riboswitch.

Overall, these results would provide strong evidence that the novel riboswitch acts as a metabolite-responsive ribozyme, exhibiting self-cleaving activity and specificity for a particular metabolite.

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