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All protein coding sequences in the iGEM Registry are supposed to end with a double stop codon. Presumably, this is to decrease the potential for read-through, which could be problematic if one is putting together a polycistronic design.

If the design is intended to have only one CDS, however, and is targeted at a prokaryote (which have "backup" ribosome release mechanisms), then is there any problem with using only a single stop codon?

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The impact of any read-through from a leaky stop codon in an expression unit with only one CDS would probably depend on a few things, mainly (i) where is the next in-frame stop codon, (ii) what are you trying to express, and (iii) how leaky is the stop codon?

In cases where the next in-frame stop codon is only a few base pairs away, there would probably be little impact, however in other cases the next stop codon could be far away. In these cases, there are two things which may cause an impact.

The first is that a long peptide sequence could be added to your protein, which depending on what you are expressing, may cause your protein to misfold or lose functionality.

The second is that you could get ribosome stalling, especially if any of the codons between your stop codon and a second stop codon require rare tRNAs. As you mentioned in your question, there are mechanisms for rescue in these scenarios, however if your CDS is expressed under a strong promoter on a high copy number plasmid, this mechanism may need to be mounted much more often than usual and cause burden on the cell. I should note that this is speculation on my part as I can’t find any studies which have shown this.

The actual impact any of these scenarios may have on your system would likely be strongly dependent on how leaky the stop codon actually is. As there are many examples of constructs which use only a singe TAA in their design with no apparent negative affects, presumably under ‘normal’ circumstances there is little risk to using a single stop codon.

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If there is only one CDS, presumably there will be a transcriptional termination directly after the CDS (after the stop codon actually), which makes any read-through coming from using a single stop codon almost negligible.

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  • $\begingroup$ Is there any issue with ribosomes getting stuck on the terminator? $\endgroup$ – jakebeal Mar 18 at 21:10
  • $\begingroup$ Actually, somethings we tend to forget in prokaryotes are: 1. in general there are many ribosomes translating at the same time, the same mRNA. 2. Ribosomes start to translate directly on the nascent mRNA, as RNAP is transcribing. $\endgroup$ – Alejandro Vignoni Mar 18 at 21:15
  • $\begingroup$ What about on the other side? Can they get stuck on the end? $\endgroup$ – jakebeal Mar 18 at 21:17
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Perhaps I could add for eukaryotic systems which terminate all stop codons by a single protein, eRF1, this study by Schmied et al., showed readthrough of all stop codons to be less than 0.2% by wild-type eRF1, which seems acceptably low! I am unsure how this would transfer to prokaryotic systems which diverges in its use of two release factors - RF1 and RF2. I would of thought termination by UAA which is shared by both factors would be quite stringent.

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