The RNA World - Before & After Michael Pearson
The RNA World began when the first RNA molecules came to possess the ability to replicate template
RNA molecules. However, there must have been chemical reactions that created ribonucleotides, and a
mechanism to polymerize RNA before this RNA world began, or it couldn't have.
I believe that the original replication ribozyme was self-complimentary. This would be an easy
stepping off point from a pre-RNA world mechanism of RNA polymerization, provided that the mechanism
included a template based step (even if the templates were randomly generated).
For example:
Step 1) Polymerization of random ribonucleotides Step 2) Polymerization using the first strand as a
template Step 3) Ends of complimentary sets are connected, forming double stranded hairpins which
cannot be used as templates Step 4) Hairpins having alternate (non-hairpin) stable conformations
could be used as template for another round of the same type of polymerization, however, they would
not be connected at completion because they would separate from each other upon switching to their
alternate conformation Step 5) Selection takes place as self-complimentary RNA strands with the
highest non-hairpin stability are kept in a replication cycle the longest
Some of these alternate-form stable strands had the enzymatic ability to polymerize RNA on a
template. Those which preferentially replicated copies of themselves, as opposed to any random
template, became most abundant. When one variety of RNA reached a certain level of specificity and
activity it could become independent of the rest of the polymerization system.
Through increased selection, the three dimensional structure at the 3' end of the replicase was
exclusively recognized for initiation of replication. The structure of this 3' replication tag (3rt)
was similar to what we know as tRNA.
As the replicase evolved higher efficiency, it was able to copy longer RNA strands with fidelity.
Because only the 3' and 5' sequences were absolutely necessary to direct further rounds of
replication, the internal sequence of some of the molecules could grow longer and change. Some of
these RNA molecules developed new functions that improved on the replication process, catalyzed the
synthesis of ribonucleotides, etc.
There were drawbacks to having a 3rt attached to these newer RNAs. This marker would have severely
limited the range of catalytic possibilities. A ribozyme developed which specifically cut off the
3rt. Because the original replicase, whose 3rt was essential to function, could not be a substrate,
cleavage specificity was mediated by a small change that did not effect replication, such as the
sequence at the cleavage point.
Without other changes, these maturing steps would have allowed new important types of RNA to be lost
as their 3rts were clipped off, preventing replication. A new ribozyme associated with the replicase
functioned to cap the 3rt of all new RNA strands randomly with one of four amino acids: glycine,
alanine, valine, or isoleucine. RNA capped with glycine or alanine (M (maturation) type amino acids)
could still be cleaved. Because of their larger and branched side chains, those capped with valine
or isoleucine (R (replication) type amino acids) could not be cleaved, and were designated for
replication only.
Before replication could begin, the R type amino acid had to be removed. It was clipped off with the
final nucleotide (A from CCA) and the removed nucleotide would be replaced by the replicase prior to
copying. After replication, another amino acid would be added.
Unlike the replicase, most of the newer functional RNAs (fRNAs) were strand specific; they had to
replicate through non-functional template strands (nftRNAs). Selection to reduce the waste and
efficiency loss associated with cleaving ends from nftRNAs changed the aminoacylation system. A new
ribozyme developed which could identify fRNAs, and cap them only with M type amino acids. Because
the replicase constrained further structural change, the strand specificity came from sequence
differences near the attachment site (i.e. in the acceptor arm). As this new ribozyme developed, the
older aminoacylation ribozyme became specific for adding only R type amino acids, but could still
attach them to both the nftRNAs and fRNAs. Together, these two ribozymes eliminated processing of
nftRNA, while ensuring that some of the fRNAs were designated for replication.
Another ribozyme developed which would cleave off the reverse compliment of the 3rt on the 5' end
(5anti-rt) of fRNAs. This built upon the ribozyme that removed the 3rt from fRNAs, and worked by a
mechanism that would couple 5anti-rt removal to 3rt removal. This complex was associated with the
replicase as well.
At this point we have a large complex housing a number of functions, here is how it would operate:
Step 1 – free subunit ‘A' specifically binds capped 3rt Step 2 – free subunit ‘B' specifically
binds 5anti-rt Step 3 – subunits ‘A' and ‘B' come together, looping the RNA so that the ‘anti-
codon' arm of the 3rt base paired with its reverse compliment sequence in the 5anti-rt ensuring
proper alignment
If RNA strand is fRNA capped with an M type amino acid:
1) Removal of 3rt by subunit ‘A'
2) If and only if 3rt becomes removed, then removal of 5anti-rt occurs
3) Release of mature fRNA from complex
4) Subunits dissociate and release cleaved pieces (one of which is an alanine or glycine capped 3rt)
If RNA strand is fRNA or nftRNA capped with an R type amino acid:
5) Cleavage of 3rt is prevented by side chain of R type amino acid
6) R type amino acid is clipped off with last nucleotide by a small ribozyme ‘C' which is part of
the larger ‘A' subunit
7) 3rt end is transferred toward the replicase (which had been loosely bound to the complex)
8) Replication commences, and the replicase moves along the template which remains held in the
complex by its 5anti-rt
9) Upon nearing completion, the replicase re-approaches the complex from the opposite side, its 3'
end interacts with the complex, causing it to dissociate and release the 5anti-rt so replication
can be completed
This system left a number of bi-products that needed to be recycled. The cleaved 3rts capped with M
type amino acids could have been used in the first cell wall peptidoglycans and cross-bridges. The R
type amino acids needed to be removed from their nucleotide before they could be used again.
Improvements to the system allowed for ways to eliminate bi-products, or efficiently recycle them.
The R type amino acid was no longer removed by simply being clipped off with a nucleotide. Instead,
once the 3rt to be replicated was bound into the replication/cleavage complex, the amino acid was
removed by having one of the free aminoacylated 3rts base-pair with the next few bases of the 5anti-
rt. The amino acid was then transferred, and replication could begin. The small ribozyme that
previously clipped off the amino acid for replication was no longer necessary for that function, so
it assumed a strictly structural role.
As before, the 5' end of the template remained bound while replication occurred and the replication
machinery moved along the template from the 3' end back toward the complex. When they came into
contact, the 3' end of the replicase (containing the original tRNA-like structure that had been
selected as the marker for replication) would function as a release factor, allowing peptidyl
hydrolysis.
This replication/cleavage complex was the protoribosome.
The bi-product of replication was a dipeptide consisting of an N-terminal valine or isoleucine
followed by glycine or alanine. The two peptides would then be cleaved apart by another ribozyme,
and the amino acids could return to the cycle.
Once the R type amino acid was removed, multiple replicases could initiate transcription of the same
strand, one after another. Because of this, the amount of cleaved aminoacylated 3rts from fRNA
outnumbered the amount of R type amino acids needing transferred for replication. This led to an
increasing amount of cleaved aminoacylated 3rts. This problem could not be corrected by a ribozyme
that cleaved that amino acid from the RNA, as this would introduce the possibility of cleaving amino
acids off 3' ends of unprocessed strands. Because a ribozyme that cleaved amino acids apart did not
have this complication, the peptide bond formation was not limited to one per strand. The reaction
could continue until there was no anticodon to match the next codon, or replication was complete.
The result was an oligopeptide that could be cleaved into individual amino acids which could return
to the cycle. This process was regulated to keep a balance in capping fRNAs for replication vs.
processing. Limiting the breakdown of oligopeptides (which, except for the first amino acid, were
composed entirely for M type amino acids) was crucial for keeping fRNAs in the replication cycle.
Although the anticodons of all the 3rts were originally the same, a number of benefits came from
adding more amino acids and anticodons into the system. The only restriction on anticodon sequence
change came from the replication requirement that anticodons of complimentary strands remain that
same. More variety lowered disruption from 3' and 5' ends from two different RNA strands binding to
the protoribosome. Also, the ability developed to control which RNAs were preferentially replicated.
For example, an RNA with a second codon requiring a rare anticodon was replicated at a lower rate
than one requiring a common anticodon.
Proteins at this point, were mainly for storage and recycling of amino acids used to tag the 3rts.
The protoribosome allowed them to be transferred form a mostly unusable state (connected to cleaved
3rts) back into a useable one (free amino acids).
Random oligopeptides could interfere with RNA reactions though, so it was advantageous for proteins
to be reliably made with a specific order of amino acids to minimize harm. Because they were
translated mainly from template strands complimentary to functional RNA molecules (with sequences
that could not freely change) it was the 3rts that had to change. However, because the amino acid
was not placed on the 3rt of fRNA based on the anticodon, it was the acceptor arm sequences that
changed. Over time, these acceptor arm sequences became associated with specific subsets of
anticodons in a way that produced acceptable oligopeptides, while minimizing coding changes arising
from sequence mutations.
Among the first oligopeptides were proteins that bound both themselves and the nftRNA 3rts,
retaining them in one location. Because they bound 3rts, RNA and other protein, these evolved into
simple elongation factors and ribosomal proteins. These proteins helped bring the 3rt of the RNA to
be replicated into the protoribosome. They also helped bring the next cleaved 3rt for the transfer.
These very first proteins likely did not have a catalytic role, but served structural or chaperone-
like roles for RNA or protein.
Because the replicase contained its own tRNA-like 3' end, variants of these first proteins
associated with it and increased its processivity. This ability, together with a new RNA ligase,
allowed a small change in genome structure. The ends of RNAs were joined, and groups of related
genes were regulated and replicated together. Only one 3rt was needed for replication, so
centralized 3rts previously used to specify both replication and non-replication were clipped out
from the longer transcripts in order to free the fRNAs. RNase P likely originated at this time to
help mature internal 3rts.
As the R type amino acids integrated into the process of making proteins, the anticodons of the
complimentary 3rts (those formerly at the 3' ends of complimentary strands) were divided. Valine and
alanine, for example, previously shared eight anticodons, which were divided based on the middle
base of the codon. This division depended on minimizing the disruption of order in the few essential
proteins in existence at the time, and the divisions were gradual as more templates became joined.
Among the first proteins to evolve with the new amino acids incorporated were the aminoacyl-tRNA
synthetases. The first two of these proteins (the first of each class) developed on complimentary
strands of a short gene. This gene contained a replication specific 3rt at both 3' ends, instead of
just at one, so neither strand was processed. Because these two proteins evolved as the genome
organization shifted, but before it was complete, they had to be compatible with the old system.
This meant that they had to be able to compete with each other in the placement of amino acids on
the 3rt of fRNAs (keeping some in the replication cycle while allowing most to be processed).
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The replicase.
The replicase, predating the rest of the RNA world, would have been different from the rest of the
RNA strands. Both strands were functional. Its 3' and 5' ends were necessary for its function. It
wouldn't have been aminoacylated. The replicase would have been lost sometime after the appearance
of a protein replicase.
The first protein replicases most likely were similar to negative strand RNA virus replicases which
use ribosomal proteins and elongation factors. Continuing to use them in replication even after the
bulk of the ribosome had been eliminated from the process.
As things shifted to a mostly protein world, the RNA replicases probably went through a time where
replication was seperated from the rest of the cell(?) in a protein core, such as in positive strand
and double stranded viruses. The purpose was to keep the genome protected. Only the coding strands
were free to interact with the ribosome.
The later emergence of DNA genomes (replicating through an RNA intermediate at first) eliminated the
need for such elaborate genome sequestering.
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The protoribosome.
Subunit ‘A' held the 3rt. This became the large subunit of the ribosome containing: 26S rRNA (which
would have clipped off the 3rt and 5anti-rt) 5S rRNA (small ribozyme ‘C' which clipped off the R
type amino acid with a few nucleotides – now serves mostly structural role)
Subunit ‘B' held the 5anti-rt. This became the small subunit of the ribosome containing: 16S rRNA
(which held the 5anti-rt for both cleavage and replication)
Replicase docked where the E site is now (a site mostly made of protein, not RNA), and finished at
the A site, acting as a release factor much like elongation factor G does now.
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tRNA.
tRNA developed from the 3rts. The first real tRNAs were the cleaved 3rts of fRNAs capped with M type
amino acids. These amino acids were at first only Gly and Ala, but expanded to at least Ser, Thr,
Asp, Asn, and His quite early in the development of the first polypeptides. These are all
aminoacylated by type II synthetases. The R type amino acids were originally only Val and Ile. This
grew to include Met somewhat early.
It is likely that a few of the amino acids were originally used as covalently attached ribozyme
coenzymes. These amino acids would likely have been His, Ser, and Arg. Before the emergence of
proteins, these would have been added after the removal of the M type amino acid and RNA end. In
some fRNAs, this cleavage would have exposed an internal acceptor arm minihelix (without a tRNA-
like structure) that would have been capped with one of these amino acids by different capping
ribozymes. Similar processes would also have attached non-amino acid coenzymes (CoA, Nicotinamide,
Flavin, etc.).
Many other amino acids were likely used early as substrates and cofactors in ribozyme reactions,
however, these amino acids were not being attached to the fRNA molecules (e.g. Glu, Gln & Cys).
Evidence:
a) tRNAs that can be aminoacylated based only upon their acceptor arm sequence: Val, Ile, Met, Gly,
Ala, Ser, His, Arg
b) the N-end rule of protein degradation: slowly degraded = Val, Met, Gly, Ala, Ser
c) viral tRNA-like aminoacylated 3' ends: Val (tymoviruses) (can be misacylated with His) His
(tobamoviruses) Tyr (brome mosaic virus) (can also be aminoacylated with His & Val)
d) amino acid usage change since last common ancestor: used less now = Val, Ile, Gly, Ala, Ser,
Thr, His, Asp, Asn used same now = Arg, Met, Pro used more now = Glu, Gln, Cys, Leu, Tyr,
Trp, Lys, Phe
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