Question

You’re working with an mRNA transcript and want to characterize precisely where initiation of translation starts and where RNA binding proteins interact. Explain the experimental approach you’ll take to do this.

Answer 1

To obtain precisely where the RNA binding proteins interact, the following experimental approach can be followed:

1. The mRNA must be transcribed and it must be divided into two test tubes.

2. In test tube 1, add only RNA and in test tube 2, add mRNA and any one of the RNA binding protein.

3. Incubate these test tubes in appropriate buffers at physiological temperatures where the RNA binding proteins bind to the mRNA.

4. Run the both the samples in two different lanes of SDS PAGE and in the third lane run only the RNA binding protein and in the 4th lane run the protein marker.

5. Perform a western blot analysis for the above formed gel and you can see the band of the RNA binding protein in the 2nd lane a bit higher than the 3rd lane.

6. Once this is confirmed, degrade the RNA in both the tubes and then run on the gel. The region of RNA where the protein is bound wont be degraded in the 2nd tube. Sequence this region and this is the RNA binding protein region in the mRNA.

To find where the translation initiation starts, sequence the whole mRNA and then perform the above experiment. Once the RNA protein binding sequence is known, the first methionine codon on mRNA after this RNA protein binding region is the place where the translation starts.

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