I recently encountered an intriguing situation while examining a plasmid constructed by someone else for a eukaryotic expression system. This plasmid contains a unique arrangement of open reading frames (ORFs) that has sparked several questions regarding the potential outcomes of their translation.
In this plasmid, there is an ORF near the 5' end, where the translation initiation site is quickly followed by a stop codon, potentially resulting in a very short peptide. More interestingly, nested within this first ORF is a second ORF that begins inside the first ORF and could potentially translate into a much longer protein, consisting of 500 amino acids.
Given the common understanding that eukaryotic transcripts typically feature a single ORF, the discovery of this arrangement has led me to ponder the following questions about the translational dynamics in this specific scenario:
Furthermore, I'm curious about how this scenario might differ if the plasmid were used in a prokaryotic system, which is known for its ability to translate multiple ORFs within a single transcript.
I'm seeking insights, experiences, or any relevant literature that could help shed light on the translational strategies employed by cells when faced with plasmids containing nested ORFs, especially in the context of eukaryotic expression systems.
Thank you in advance for sharing your knowledge and experiences.
There are a myriad examples of this sort of ORF structure in eukaryotic viral systems (particularly RNA ones), where it has evolved to diversify/expand coding capacity and/or regulate viral gene expression. At first pass you can predict relative expression levels by examining the Kozak consensus around each AUG codon. We usually consider the -3 and +4 positions to categorise them as strong, middling and weak. If the first AUG is strong, you typically won't get much of the second product. If the first AUG is medium or weak and the second is strong, you'll get lots of the second product. Confounders to look out for; 5'-UTR length and protein stability. AUGs very close to the 5'-cap are often not translated well. Shorter proteins are harder to detect and if they're too short (or the wrong sequence) to fold up into a coherent structure, they'll be turned over rapidly and can be hard to detect unless (or even if) the proteasome is turned off.
After that, it can get properly complicated with all sorts of ribosomal gymnastics depending on the exact sequence of the transcript.
Bugs - need to consider Shine-Dalgarno (sp?) sequences or lack of, I think. But my undergrad lectures on this were many years ago :)
Cheers
Paul
I think the answer Paul Digard gave is good for what will happen in eukaryotic system in most cases. With regard to bacterial expression, you can have many AUGs and they can all be used, but how well is going to depend upon the quality of a ribosome binding site (Shine-Dalgarno) that is appropriately positioned.
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