Question

You are studying a human mRNA that you believe is controlled at the level of translation. You think that the 5’-UTR plays a role by binding to a protein that prevents the initiation of translation. To definitively determine the role of the 5’-UTR, you decide to clone the 5’UTR of the gene upstream of GFP open reading frame in a mammalian expression vector.

Question: Describe an experiment that you would perform using this vector to prove that the 5’ UTR plays a role in the control of translation; explain the result of the experiment would be if your initial hypothesis is correct.

Answer 1

It is given that a given sequence element located in the 5'-UTR is required for activation of translation.
To confirm this, clone the sequence along with a reporter gene such as GFP and transform into bacterial cells.
We have to use two constructs.
In the first construct, put the WT 5'-UTR sequence along with the GFP
In the first construct, put the mutant version of the 5'-UTR sequence along with the GFP

If 5-UTR is indeed required for translational activation, we would observe high GFP signal from the cells that are transfected with the WT construct.
Otherwise, there would not be any difference in GFP signal from both constructs.