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Proposal for experiments about the origin of RNA replication

Tym Lawrence tym at unixg.ubc.ca
Thu Nov 23 04:19:04 EST 1995

Hello, this is a outline for a proprosal I am starting for a UBC class 
evolutionary genetics- any comments or feed backs should be mailed 
dirctly to me at TYM at UNIXG.UBC.CA - so thanks, and have a good one (or two).


The broad question I want to answer is :


in other words I want to explore how the first RNA, or pre-RNA (perhaps 
in a PNA / backbone form as suggested by Eghlolm (1993), Neilsen (1991) 
or Wittung et al (1994)) molecules became SELF-REPLICATING and then began 
to evolve to allow other RNA molecules to replicate.

This is of course a broad and complex question, with so many variables, 
possibilities and unknowns that it seems impossible to answer. 

You canUt just put into a vat all the nucleotides, amino acids, and other 
raw materials... and see if you can eventuate with a self replicating 
molecule, or a molecule that will act as a polymerase for other RNA 
molecules. What problems are ther that need to be overcome ?

Firstly: What basic units are needed? Do you need: pre-biotic chemicals 
(like the ones in Stanley MillerUs reaction vessel such as H2O, HCN etc), 
activated nucleotides, amino acids or some simple polypeptides, peptide 
nucleic acids, aminoacyladenylates, phosphates, activated phosphates 
(ATPUs ?), metal ions (Zn, Mg, Ca, Fe ?) and transition elements1 Then 
there are all the possible derivatives of these items (eg glycinamide2)

Then, how to mix these elements: In what concentrations? With what 
pressure3? Wtth what sort of buffer solutions? Plus what sort of surface 
should they have access to (eg clays or mineral sands),

Still even with all these elements in the right proportions, it is likely 
that random chance could only generate up polymers perhaps up to the 
thousand bits of information level (ie one Kilobyte). Eventually one of 
these molecules had replicative ability (either replicating itself and/or 
itUs favoured neighbours). With ligation ribozyme, this eventually lead 
to the most primative ribo-organism which probably had over one millions 
of bits of information (one Mb)  

We would also have to overcome problems of Pyramidine to Purine template 
directed replication4 and also address issues of homochirality5 (see 
below about these two issues)

Also, would the RNA/PNA template and replicating molecule for a double or 
triple stranded structure6 ? Finally, there is much to support the theory 
that replication of informational molecules was not self contained, but 
involved co-evolution of multiple genetic systems7

The way that I see it, is that PNAs, RNAs, most Amino Acids, and peptides 
up to hundreds of units long8 are able to be generated by non-enzymatic 
pre-biotic pathways, and these molecules, although not be very big, are 
still quite stable.  The likelihood is that these molecules formed on 
mineral sands, such as montmorillonite.. and those that had emzymatic 
functions were useful, and thus retained. However, to survive, and 
increase, replication is necessary. This could happen in two ways:

a) a molecule acts as its own replicative enzyme. It is thus self 
replicating. This type of pathway has not yet been demonstrated fully in 
an RNA world, but many of the pieces of the puzzle are present: eg RNA 
can assemble activated mononucleotides to form the complementary of the 
purine rich strand9.. and now other experiments have shown that there are 
ways around the problem of forming the purine rich strand from the 
pyramadine rich strand. 

b) if a molecule forms that aids (catalyses) these reactions, thus 
increaing the rate of template directed nucleic acid formation, then it 
could aid in the formation and propagation of other molecules. This 
RNA-world polymerase could be a Ribozyme, or a combination of RNA, PNA 
and Amino acids. This molecule, a RNA polymerase, would be a keystone for 
the RNA world.

Thus the narrowest query that leads directly from the main question is to 
is there a Ribozyme or other RPre-protein worldS polymerase that can 
catalyse the replication/self replication of RNA or PNA molecules.

Would the issue of homochirality cause a problem- probably not, because 
the first RNA polymerase to form would have a preference for binding to 
either left or right handed templates.. meaning that the first one to 
form (ie a left handed RNA polymerase) would then define the chirality 
that was to survive and thrive. - Ie the molecule that I want to find 
would explain homochirality.

NOW - How to find this molecule: 

A)  Look in Genebank for sequences that show great homology between other 
polymerases. We could then use this as a start point for RNA synthesis 
and experimentation.

B) Use the methodogy of creating randomized DNA sequences with similarity 
to very basic polymerases, then produce RNA from these, and assay them to 
try for a RNA polymerase.

C) Using the methods of in vitro selection and evolution of new RNA 
catalysts10 which have recently provided self aminoacytlating RNAUs 
(amongst other de novo RNA cataylsts) we could then evolve and mutate a 
RNA polymerase to get a molecule that can catalyse the replication of the 
earliest codes of life.

BUT - as a one year old article said - finding these new Ribiozymes is 
RGuesswork and Randomness (for now at least)S11 and to find these types 
of molecules must also be the goal of many other labs.. so either a more 
specific query, or a novel approach is need. So, any suggestions, from 
any quarter, would be appreciated.

Tim Lawrence.

1Okihana 1984
2Yanagawa 1984 looked at glycine polymers
3Nickerson KW 1984- discussed the effect of pressure, water activity and 
polymer formation
4 Li & Nicholaou 1994 have done work on this
5I think Stanley Miller has done work on this
6Li and Nicholaou 1994 have worked on this.
7 see Bhler et al, Nature Vol376, August 1995 for information on the 
interactions between PNA and RNA.
8Getz WM 1990
9 Orgel 1993
10 Joyce 1994 and Chapman 1994
11 Nature vol 372, 3rd November 1994, p30

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