We did an investigation to see if translation was initiating at another ATG start codon due to leaky scanning. We constructed a plasmid expression vector and a restriction digest to confirm the plasmid and to see which initiation codon was being used.
We found, by an in silico investigation, that possibly initiation was caused by leaky scanning and found the predicted product of the protein domain. Molecular weight predictions implied that the protein produced was from the second ATG.
Sequencing alignment showed that there was not much conservation with other vertebrates.
Could future work consist of mutational study to knockout the first ATG and see that if the same, smaller protein is produced, it will confirm that initiation did indeed start at the second ATG?
What possible other things could I do to further improve the project in your opinion?
Hi Ritesh,
A couple things...
1) Is the product of the second AUG in the same frame as the first i.e. is it a truncation of the normal protein or a completely different product. If they are different you could make antibodies to the two separate products and confirm their identity by Western. Even if it is a truncation you could make antibodies to the 'extension' of the normal product and see if that reacts with the longer product and not the shorter one.
2) If it is leaky scanning, there should be an effect of the context of the AUGs of each ORF according to the Kozak consensus. So, you can mutate out your AUGs (i.e. mutate the first one and see if more initiation occurs on the second, mutate out the second and see if you now fail to get a product). Importantly, in addition to this make mutations of the +4 and -3 nucleotides of the AUG to alter the Kozak consensus and see if this changes the ratios of the two products. The scope for changing the context is quite large and will certainly add weight to your findings.
These are some good things to start off with, I highly recommend the papers of Marilyn Kozak, they may give you some more good ideas (if I remember, most of these were looking at proinsulin RNAs).
Hope that helps :)
Obviously attempting to work out the function of the second product is a good one too! But the other two approaches are simpler and will get you started.
many thanks for your advice, Mike, i will definitely look into the points you made here and look at Kozak's papers.
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