Redesigning the ribosome: chemical goals

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The ribosome is an enormous, complicated RNA machine. We are asking you, intrepid players, to stabilize it. We've made a lot of noise about how this could enable new chemistry. But why is that? I wanted to give a brief, no-background-necessary sketch of the chemistry and physics underlying why that is.

Enzymes (whether made of protein or RNA) are a type of catalyst. A catalyst lowers the "activation energy" of a reaction, which allows it to go faster. (Properly, "happen more often"; it's not like bond breaking and forming can be truly 'sped up', but rather that particles bump into each other all the time but usually nothing happens.)

How can a catalyst lower a reaction's activation energy? Two points. First, "activation energy" is made out of two components: the "delta-H," or change in "enthalpy," and (temperature times) the "delta-S," or change in "entropy." In order to lower a reaction's activation energy, it either has to make the change in enthalpy more favorable or it has to make the change in entropy more favorable. (It can do both, but generally speaking, catalysts do one or the other.)

OK, so a catalyst either makes the change in enthalpy more favorable or the change in entropy more favorable. The enthalpy of a particular molecular state is (essentially, for our purposes) its internal energy: the energy of its bonds, the nice intermolecular forces it's experiencing, etc.) The entropy is a little more complicated because it's a measure of the disorder of a system. In some sense, it's a way of saying "how specifically you have described the system." If you say "two molecules, within a really huge box," that is a very high-entropy state (high disorder) compared to "two molecules, RIGHT NEXT TO EACH OTHER."

So first, let's compare each of these quantities for a bond-forming reaction like the formation of a peptide bond. Enthalpically speaking, the transition state is terrible. All the bonds that are going to break during the course of the reaction are "half-broken" and all the bonds that are going to form are "half-formed." Colloquially, we are getting none of the benefit and all of the pain. It's the worst. So if you could form nice interactions with the transition state, that would make the terrible, costly delta-H a little better. A lot of catalysts do just this: many reactions, especially in biology, proceed via acid-base catalysis where one group donates a proton to another, making it a better leaving group. (Look up serine proteases for an example.) Entropically, the transition state is also terrible! Remember my example of a low entropy state? In order to form all those bonds, you have to take two molecules that were once wild and free and force them right next to each other for as long as the reaction takes.

So, here's the big kicker: if the ribosome were reducing the delta-H of the reaction by stabilizing the transition state, that would be a bit frustrating for us because you'd need intricately different ribosomes for EVERY reaction. But that's not what it does! The ribosome reduces the delta-S of the reaction by DESTABILIZING the reactants. That's right: entropically, the transition state is hopeless -- both reagents are constrained -- so the ribosome makes the entropy of the starting materials worse by forcing them together (they're chemically bonded to tRNAs, and the ribosome binds tRNAs very tightly). This is a very general mechanism -- I didn't have to tell you anything about the identity of the reactants -- so we suspect that the ribosome could be made to function as a general, programmable synthesis device if we figure out how to accommodate each reagent in other places. (For example, there is an "exit tunnel" where the growing polymer chain gets extruded, like those dolls with play-doh for hair. If your monomer is too bulky for that tunnel, bad news.)

And if we can make the ribosome REALLY stable, we can play around with these critical areas and start to hollow them out if we need to, without risking the whole ribosome falling apart.

Thanks for reading! Let me know if there's anything I can clean up to make this a clearer introduction to our theory.
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andy.watkins2, Researcher

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Posted 5 months ago

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Sarah Smaga, Researcher

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Hi EteRNA players!  If there is background information about the Ribosome Pilot challenge that you were unable to find elsewhere (or wasn't very clear), let me know! Just send me an email (sarah@gem-net.net) and I'll ask one of our researchers to put something together.  

C-GEM has a blog and we'd love for it to be an additional resource for players. Right now, the blog is mostly about developments in the literature, but we're looking to expand into explainer articles (and hopefully videos and animations after that). 
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JR

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Andy said: " We are asking you, intrepid players, to stabilize it."
What tests will be used to judge whether our designs have "stabilized" the Ribosome?

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andy.watkins2, Researcher

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So, tests of stability per se won't be part of the first round: on the experimental side, we'll settle for "mutated, but still functional." Eventually, we could test traits like thermostability (does it still function at higher temperatures?), or the ability to fold without so many proteins around (the ribosomal proteins help it fold), and so on -- but that's longer term. We just haven't explored much of the mutation space of the ribosome so we are interested right now in seeing what mutations are possible at all without breaking it, and using the secondary structure ensemble energy as a proxy for whether we're probably making it a little more stable in at least one sense.
(Edited)
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DigitalEmbrace

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Sarah, I sent you an email on June 18. Did you receive it?
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Gerry Smith

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Is there a ribosome that does two things that we could try to change so it only does one?  ...to help figure out what areas relate to function?
(Edited)
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andy.watkins2, Researcher

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To our knowledge, all natural ribosomes catalyze one reaction: the addition of a single amino acid to the C-terminus of a growing peptide chain. But in principle that's a really good idea: if we were to happen upon a ribosome that could do two things [and we're really interested in designing IN a third activity], it might be very valuable to test our understanding of HOW it's doing those two things by trying to 'shut off' each of them.

If you're interested more about what areas relate to function, you'll be especially interested in the Domain V puzzle for the 23S. Domain V contains the peptidyl transferase center -- it's not where ALL of the magic happens, but it's where most of it does.
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Gerry Smith

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In article Rhiju shared, "Modular Assembly of the Bacterial Large Ribosomal Subunit" it says "the late maturation of the PTC has been observed in the distantly related B subtilis previously, suggesting that this late timing is a conserved feature of the modern ribosome..."

Are there any maturation patterns areas of what we are currently working on that we could use as probabilistic guides to both place and avoid mutations in?  To see if grouping mutations in these areas either improve or hurt?
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Gerry Smith

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Re mutation guidance from maturation patterns:

 Just as eterna is now using IUPAC codes to more efficiently guide mutation patterns, how can we use observed conformational stages/motif pattern sequences to do the same?

 both Rhiju's article and the one Omei recently shared, (see below), point this direction.

“A metastable rRNA junction essential for bacterial 30S biogenesis” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6007441/

   
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DigitalEmbrace

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Thank you, Andy. These explanations help us amateurs understand the science a bit more. In your short introduction to the Ribosome Challenge, you stated the goal is to improve the energy gap between target structure and MFE, using as few mutations as possible. How few mutations to 16S and how few mutations to 23S would you guess, ideally? Are there any research findings relevant to how many mutations can be made?
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spvincent

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A slight tangent but there's a very readable book about the efforts to determine the structure of the ribosome called "Gene Machine" by Venki Ramakrishnan (he and a couple of other researchers shared the Nobel Prize in chemistry in 2009 for this work) .

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Gerry Smith

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"Gene Machine" was readable and fascinating!  Thanks for this recommendation.  So helpful in background info for eterna labs too.
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DigitalEmbrace

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I'm 2/3rds through Gene Machine and learning a lot. Had been looking for a book explaining recent developments in ribosome structure and function.