Proposal for experiments about the origin of RNA replication
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 :
HOW DID THE FIRST TEMPLATE DIRECTED RNA/Pre-RNA REPLICATON BEGIN ?
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
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.
2Yanagawa 1984 looked at glycine polymers
3Nickerson KW 1984- discussed the effect of pressure, water activity and
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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