Sunday, January 08, 2017

Pondering What Is Lost In Teaching Translation

I'm good at acquiring distractions, and a relatively new one is Quora.  This site allows users to ask questions which are then answered by members of the community.  I lurk in a number of fields, but have answered a few questions related to genomics and related fields of biology.  Tackling a question last night required re-learning some details I was disappointed I had forgotten.  In researching to regain that knowledge, I skimmed a number of study guides online, which leads to this post.
Questions on Quora can be just about anything, but I only consider writing answers for a small number.  Some ask purely philosophical questions or downright bizarre science fiction related questions (along the lines of "If aliens really do exist on Omicron Persei Eight, would NotI be a rare restriction site in their genomes") or typical creationist trolling  ; those I don't touch.  There unfortunately aren't "you really need to do your own homework" or "Wikipedia answers that in seconds" buttons, which really could be useful.  So those are out.  Plus often someone else has written something, so if that's pretty good I tend not to write an answer, though sometimes I leave a comment or use the "suggest a correction" function for a fix.

So what does spur me to write something?  One class is an abundant one, people asking "how can I get into computational biology as an X?", where X could be "a computer science major" or "I haven't picked a major yet" or "long term tech worker".  At some point I should just write a blog post on that to point all of these too, though each deserves customization.  

A second class is when the question is interesting but the answers are not.  Wrong answers or tautologies are just irritating, so getting a good answer in the mix feels good.

A final general class that gets me going is when the question is touching on something particularly deep or amazing about biology.  Questions whose answers illustrate general themes of biologg.  If it makes me going fishing to answer, all the better, even if it means admitting I've forgotten something I once learned.

This recent question fell into the last two categories.  The question is "Why is a protein synthesized from its N-terminal end to its C-terminal end? Is there any specific reason?".  I was annoyed I couldn't just rattle off a good answer, but worse the two prior answers were not much help .  One reiterated the nature of a peptide bond and then gave an explicit "I don't know" for the answer to the question.  The others simply stated that protein synthesis starts at the N-terminus and ends at the C-terminus, though one hinted at a question of energetics.

So off I went in Google to try to find the answer, and it was surprisingly hard to dig up.  After many rounds of going through documents on translation elongation, I finally found the answer. It's roughly along the lines I remembered, but now I have much better context to understand it.  But in all that digging, I realized that many of the guides are seriously flawed, even for the level of instruction they appear to be aimed at.

In the ribosome, both the nascent polypeptide chain and the incoming amino acid are linked to tRNAs via a phosphoester linkage.  That linkage is installed by the tRNA synthetase.  The amino group on a an amino acid can make a nucleophilic attack on the phosphoester linkage.  The question is which amino group makes the attack on the opposite ester, as at first glance there is an apparent symmetry.  

The answer is that it is the incoming amino group in the A site which attacks the phosphoester linkage of the nascent polypeptide chain in the P site.  In this way, it is simple to put the two reactive groups in close proximity, albeit requiring translocation of the peptide chain each time from the A site back to the P site.  Imagine the converse chemistry; the amino group of the nascent chain would get farther and farther away from the linkage of that chain to the tRNA.  This would necessitate the peptidyl transferase active site to be on some sort of extendable linker.  Nature picked the much simpler scheme.

After finding the answer, I started thinking about the diversity of study guides I had dug through to get to my result.  A few had downright wrong information; one I saw talked about hydrolysis of the aminoacyl-tRNA linkages during elongation; that would be disaster.  But many were simply missing many of the details.  

Now mRNA translation is central to biology, so it is taught many times in increasing detail.  TNGs short biology unit in middle school, for example, mentioned ribosomes and said they make proteins, but that was about it.  His biology class last year brought in topics of tRNAs, mRNAs and codons, but not a lot more.  At Delaware, I remember some treatment in freshman bio and more detailed treatment in biochemistry; there could well have been other goes at it in microbiology, molecular biology, cell biology and genetics.

I'm not performing a rigorous analysis of these guides; more a series of anecdotes.  It would be a neat advanced undergraduate assignment to have each student take 3-4 such guides and compare them for content and accuracy; in a course on teaching biology I could see looking at this issue for different topics as a recurring theme.   Different guides were clearly aimed at different levels of instruction, ranging from intro classes to very detailed upper level classes.  Some of these advanced guides and outlines went into the translation cycle in deep detail, particularly the cycling of elongation factors.

With regard to the level of detail to be taught for a given type or level of class, there are really two questions.  First is how much detail to go into on the canonical process. Given that we understand the process of exquisite atomic detail, there clearly must be some filtering.   But also is important to decide how many exceptions to point out, as this is biology and the exceptions sometimes seem to overwhelm the rules.  I love getting into the exceptions, as they often illustrate subthemes, but others have different tastes.

A surprising omission from most of the guides I saw is the fact that the peptidyl transferase activity of the ribosome is catalyzed by ribosomal RNA; the ribosome is a ribozyme.  I feel that once you've introduced the idea of enzymes to students, then this is a topic worth covering.  Should some students start pondering the fact that while most enzymes are proteins, the obviously ancient process of making proteins is catalyzed by RNA, then a really big win is gained.

A number of the guides are completely vague on the biochemistry.  A key concept for me is that the tRNA synthetase effectively drives the peptidyl transferase reaction; this detail is missing from most of the guides I found.  This coupling of a nucleotide to a moiety to be added is a general theme.  For example, glycosylation is enabled by first coupling the sugar to a nucleotide.  It would be worth noting at this juncture also that there exist biological systems which generate polypeptides without mRNA, tRNA or ribosomes -- but these "non-ribosomal peptide synthetases" still use the logic of first coupling an amino acid to a nucleotide.  Similarly, the abstract logic of the ribosome is important, in which an incoming subunit attacks the extending chain when both are bound to distinct sites on the enzyme, followed by resetting the chain to its prior location.  This same logic shows up in fatty acid synthetases and polyketide synthetases.

Sticking with the tRNA synthetase theme, many of the guides (but not all) mentioned the issue of correct selection of amino acids by tRNA synthetases being crucial to the fidelity of the overall translation process.  Unmentioned by any, though, is the fact that this accuracy is boosted by proofreading of aminoacyl-tRNAs before they are released from the synthetase.  Incorrect chargings are released by hydrolysis, generally by an additional site on the same enzyme but in a few cases by stand-alone enzymes.  That's certainly a college-level topic, but an important one.  Proofreading of prior events is a recurrent theme across biochemistry, and so important to highlight.

Virtually every guide stated there are 20 amino acids conjugated to tRNAs and 20 different tRNA synthetases (in two structural classes).  Well, those statements are often not strictly true.  The exceptions to this are fascinating, but are a question of detail.  I do think the fact that selenocysteine and pyrrolysine are incorporated by tRNAs is worth introducing, though they bring with them the extra baggage (also in my opinion worth it) of explaining that their tRNAs are translated in a manner dependent on context around the cognate codon.  But there's also a fascinating exception on the pairing of tRNA synthetases and amino acids and tRNAs.  Some genetic systems lack glutamine tRNA synthetases, instead charging tRNAGln with glutamate and then a separate enzyme amidating the glutamate.  Again, probably something to reserve for upper level classes, but worth covering.

Some, but probably not most, of the guides mentioned alternative genetic codes.  Since we all carry one around in our mitochondria, I feel this is important to mention.  Perhaps the most important reason is to simply stress that one of the most central and ancient processes in biology is riddled with exceptions.  Some students will be turned off by this lack of rigidity, but I'm sure others (like myself) will thrill to the complexity.

Well, that was my journey back into the details of ribosomal translation of mRNA and what details I feel should not be omitted from instruction but apparently often are, triggered by a dissatisfaction with the answers to a question on Quora.  

1 comment: said...

I see, the ignored question of biology -

Blame it on two lightweight scientists Watson and Crick.