SEP V020: Recommend use of polypeptide region for 2A Sequences

[jakebeal]

This SEP proposes to add the new SO term for 2A sequences to the Protein Cleaveage Site glyph.

Read the full SEP at: https://github.com/SynBioDex/SBOL-visual/blob/master/SEPs/SEP_V020.md

[graik]

2A is mechanistically completely unrelated to protease cleavage. The SO term of "self-cleaving peptide" is in fact also wrong (and should IMO be corrected). 2A sites work at the level of mRNA sequence/structure and make the ribosome "skip", that is the ribosome releases the peptide chain it is busy translating but does not detach. Instead, it resumes translation a couple of codons further down, at a defined position after the 2A sequence.

I would advise not to connect this term to protease cleavage. There is no protease involved, it is not a post-translational processing step, and the two protein halves cannot exist fused in order to be cleaved later. Instead there is 2A-specific considerations such as: (1) it only works in eukaryotes (I think with some host-specificity as well), (2) different 2A sites have different efficiencies in different contexts (some close to 100% others not so).

It would be good to have a symbol for 2A sites but it should be different from protease cleavage.

[jakebeal]

I would not say we are connecting the term to protease cleavage: rather, this is recognizing that "protease cleavage site" was always too specific a term for the general notion of a cleavage site that is captured by this glyph.

With regards to naming: the world does tend to call it "self-cleaving" even if that's not the mechanism, and indeed the mechanism appears to not be completely settled. The SO term does indeed have an alternate name of just "2A polypeptide region" if you prefer, and leaves the mechanism undefined.

[graik]

Well, I am not too concerned about the SO term. But from a protein biochemist perspective: protease site != 2A sequence This is not some nit-picking detail. It's comparing apples and oranges.

…

On Tue, Oct 22, 2019, 21:23 Jacob Beal ***@***.***> wrote: I would not say we are connecting the term to protease cleavage: rather, this is recognizing that "protease cleavage site" was always too specific a term for the general notion of a cleavage site that is captured by this glyph. With regards to naming: the world does tend to call it "self-cleaving" <https://en.wikipedia.org/wiki/2A_self-cleaving_peptides> even if that's not the mechanism, and indeed the mechanism appears to not be completely settled. The SO term does indeed have an alternate name of just "2A polypeptide region" if you prefer, and leaves the mechanism undefined. — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#78?email_source=notifications&email_token=AAOGZXPVFQYEP2CONVJUKKLQP5AJDA5CNFSM4JDRIQB2YY3PNVWWK3TUL52HS4DFVREXG43VMVBW63LNMVXHJKTDN5WW2ZLOORPWSZGOEB6W6XA#issuecomment-545091420>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AAOGZXNNZPICARGLKZWHTE3QP5AJDANCNFSM4JDRIQBQ> .

[jakebeal]

Absolutely agreed. However, in prior discussion (#70), the consensus appeared to be that the glyph was for fruit, and that both apples and oranges were fruit.

[graik]

Reading up on that discussion, it was a rather strained consensus... the discussion started out with the same observation that 2A and protease cleavage are not the same. They may end up in a similar result but only under the condition that (i) the protease site is used in the presence of a matching protease and (ii) that the 2A site is used in a matching host.

But as an engineering feature, they are quite different:

• 2A sites are used to emulate poly-cistronic expression in eukaryotes. This is not a typical usage scenario for protease sites.
• protease sites are routinely used for cleaving off protein purification or other tags (after purification). 2A sites cannot be used for anything like it.
• protease sites can become hidden / inaccessible by protein 3D structure, 2A sites are not affected by protein structure
• protease sites are a protein feature. 2A sites are a (transcription/)translation feature

Given that both features are widely used, but each in different contexts and for different purposes, they both deserve their own symbol.

I for sure would want to know wether there is a 2A site in a design (implying separate proteins from the start, implying this design will only work in a eukaryotic context) and wouldn't want to see this confused with a protease site.

[jakebeal]

I would happily support separate development of a pair of more specific glyphs. That option for distinguishing would be very analogous to how the DNA Cleavage Site glyph relates to the glyphs for blunt restriction sites and sticky restriction sites.

Per the request from the editors for making SEPs atomic, however, I would suggest that those be developed in a separate thread.

[chofski]

I agree with Raik in this instance and think that conceptually the two elements are quite different in their function and so using the same glyph could be confusing. Would it not be better to draw such elements with two separate CDSs (which they are as the 2A sequencing is not protein coding), and use either an 'Engineered Region' or new glyph between these to denote that this region is not protein coding? That seems clearer and more accurate to me.

[jakebeal]

I definitely agree on the two different CDSs in any case, but if we're going to have a new 2A glyph, would somebody like to propose one?

[chofski]

For a paper where we considered translational frameshifting (which is different, as the whole sequence is still protein), we opted for an arrow connecting one frame to another (see Fig 4 in https://www.embopress.org/doi/10.15252/msb.20188719). This could be adapted for your case maybe as follows. This would also connect with the idea of a RBS and translation initiation due to its similarity.

2A.pdf

[jakebeal]

Interesting! Would that be better used for frameshifting, though?

[chofski]

For a frameshift the CDSs would be overlapping so I think there would be a clear difference. I'm not strongly advocated the arrow, but do think it nicely shows the skip along the transcript the sequence induces.