hi, I have an simple question related to IRES.
Regard with my cloning experiment, I noticed one thing.
promoter-gene1-IRES-reporter gene
Here is a sequence which I used. In that case, I created a control with no insertion of gene1. Consequently, gfp of control group is much brighter than that of interted gene1(overexpressed group).
I already know downstream of IRES (gene2) should be lower expression than gene1, however, what happen to this work?
As you understand, the IRES is a weak ribosome binding site (RBS). Your vector would drive gene1 with a strong RBS, such as Kozak's sequence.
If you have a gene1: The ribosome binds to the strong RBS, scans until it finds gene1's start codon, translates gene1, and terminates when it reaches gene1's stop codon. Only the weak IRES drives GFP.
If you delete gene1: The ribosome binds to the strong RBS. There's nothing to stop it until it finds GFP's start codon. Now both the strong RBS and the IRES drive GFP.
To bring GFP expression down, you'd need to delete gene1's strong RBS, not just gene1. Alternatively consider using a mutated, inactive version of gene1, as the most relevant control.
In addition to John's answer, I would also suggest that the control construct might have a higher transcript copy number in the cell since it's a shorter construct. This could then be translating into more gfp molecules than would exist in the gene1 version and, hence, brighter cells.
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