How early life copied itself

24 August 2018

Interview with 

Phil Holliger, MRC Laboratory of Molecular Biology, Cambridge

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Lava field at Kilauea Volcano, Hawaii

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Arguably one of the most important questions in biology is how life got started on Earth. A popular theory is that it began with DNA’s single stranded molecular relative “RNA”. RNA can carry genetic information but it’s also capable of folding itself into complex three dimensional shapes that also endow it with enzymic - or catalytic - capabilities. The problem is how to make a tangled 3d structure copy itself. DNA and modern RNA systems do it by using proteins to temporarily straighten out the strands to make them readable, but early life wouldn’t have had those proteins. So how did it do it? Chris Smith heard what Phil Holliger has discovered…

Phil Holliger - So in present day biology, DNA stores information in the nucleus; proteins are the sort of bricks and mortar and the engine of the cell; and RNA is like a carrier of information from the information store to the machinery - to the proteins. But I think there's compelling evidence to suggest that early life must have been a lot simpler. So there must have been a primordial biology, which may have been based mainly on RNA, and that really raises to question how these RNA strands were copied in that primordial biology because in present biology both RNA and DNA strands are copied by proteins. So in that early biology RNA would have had to be able to copy itself.

Chris - There is a precedent for RNA having enzymic - and catalytic - function though isn't there, because there are things like ribozymes, which are machines in cells where they exploit the fact that RNA has a structure and it can guide chemical reactions; so there is a sort of precedent for having that view...

Phil Holliger - Exactly, so this is actually one of the reasons why people think that early biology might have been based on RNA that, unlike DNA which is more like an informational string, as a molecule RNA has this amazing capacity to both be an informational string and fold up into intricate three dimensional shapes which can have catalytic function including the capacity to actually copy other RNA strands.

Chris - So what's the problem with the hypothesis then, if RNA can do that it can be both informational and it can be biologically active as a catalyst, why couldn't early life of just use that?

Phil Holliger - When we were trying to build such a system where RNA could copy itself, we hit upon a snag. At the ribozyme, although it could copy RNA strands very well, if they were unfolded - if they were strings - once they had assumed a three dimensional shape they could no longer be copied. So you essentially hit a paradox where RNA needs to fold up in to a three dimensional shape to be able to copy itself, but once it has folded up it can no longer be copied! The paper is about how we have begun to find a solution to this paradox.

Chris - How did you?

Phil - The idea was the following: so maybe what we needed to do is to move away from trying to build something that was closely analogous to present day biology. In modern day biology, when DNA and RNA are copied they're copied in single letter steps. So single DNA or RNA letters are incorporated one by one by the copying enzymes. But our original ribozyme had exactly the same type of function like this - it would copy RNA in these single letter steps. But then when it when it hit a structure, kind of, it could not move forward. The solution we found is that if we move to three letters step - a triplet as we call it - and this is not used in nature at all to copy the DNA or RNA - when we move to triplets, this triplets - by virtue of binding much tighter to the RNA strand - they could actually straighten out the RNA template and as we approached a structure begin to invade and unravel the structure. And this way we found that eventually we could copy even extremely stable RNA structures - structures that have a melting temperature close to the boiling point of water.

Chris - So to put this another way it's almost like you've got a sort of a shirt with lots of wrinkles in it; and it goes through the iron and gets flattened out...

Phil Holliger - Yeah... 

Chris - ...while it's momentarily flattened out, your enzyme can read that nice flat surface and make a parallel equivalent, or a copy, of that bit of the shirt and then it falls off the other side of the ironing board and gets all its wrinkles back again - so it goes back to its three dimensional shape, but you've got a copy in the meantime?

Phil - that's approximately right; unfortunately it doesn't actually wrinkle back -  I think it will stay as a double strand, because that's probably the most stable form. So in fact this is one of the problems that we are tackling now; to set the system back to its beginning, we will now have to find a way to unravel that double strand again.

Chris - So you've solved one problem and created another!

Phil - Well I think that's probably always there. I think that we always were aware that that was part of the replication cycle. Now, in the cell kind of like there are some intricate molecular machines which have exactly that function to unzip double strands. We will have to find an alternative solution, but we're working on it!

Chris - Does this mean though, that in Darwin's little pool where there must have been these early forms of life if they were using RNA the way you speculate they were, that the supply of molecules that they must have had to feed their RNA replication machinery must have been these triplets - letters three letters - joined together in a sequence?

Phil Holliger - I think the most likely scenario is that you know there was a whole you know mix of letters there: there were probably single letters, double, triple letters and maybe some quadruple, quintuple letters. Presumably the longer, more complicated letters were rarer; presumably the copying enzyme would use whatever as appropriate. So you know and a nice kind of extended strand, you know, you could probably quite happily use the single letters; but then once it hits a structure then more extended letters - the triplets etc - kind of become more useful.

Chris - How did you make the discovery in the first place though. Was this just brute force - trying loads and loads of variants of the RNA system - until it worked?

Phil Holliger - We do not have currently the molecular understanding of RNA to do bespoke engineering of let's say designed catalytic activity of copying function of exactly what we want. So what we use is an evolutionary process where we force the RNA to begin to use the substrates that we like it to use - like the triplets - by iterative cycles off of evolution in the test tube and this is a very very powerful process.

Chris - So if you've got this right., what are the implications for early life about four billion years ago?

Phil - So potentially there is really no conceptual problems with building such a system from scratch that could copy itself and with self replication presumably the copying process would make a few errors. So you have mutation as well and with self replication and mutation you would have evolution and that would really get the ball rolling towards ever more efficient self replication and you could see how that process would take off and lead to more and more complex things...

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