Mol. Cells , Vol. 9, No.5 , pp. 459-463
Molecules
and
Cells
Millireview
© Springer-Verlag 1999
Ribozymes -
Why So Many, Why So Few?
Francis J. Schmidt
Department of Biochemistry, University of Missouri-Columbia, Columbia MO 652 12 USA.
(Received on May 13, 1999)
The RNA world scenario posits the existence of
catalytic and genetic networks whose reactions are
catalyzed by RNAs. Substantial progress has been
made in recent years in the selection of RNA catalysts
by SELEX, thus verifying one prediction of the model.
However, many selected catalysts are long molecules,
leading to a question of whether they could have been
synthesized by a primitive replicator. It is proposed that
the efficiency of some small ribozymes may have been
augmented by other RNAs acting as transactivators.
Introduction
The di scovery of catalytic RNA nearly twenty years ago
was hail ed as a way to solve a probl em inherent in the
Central Dogma of Molecul ar Biology - DNA makes RNA
makes protei n. Biological informati on transfer, like all
biological processes, must have ari sen through evolution
by natural selecti on. Natural selection is a feedback system
(Seaborg, 1999) - the level of fitness conferred by the
phenotype allows for the diffe renti al reproduction of the
genome specify ing that phenotype. Thi s is not a problem
fo r a cell , since the genotype and phenotype are bounded
within a membrane that defines the organi sm. But what if
there is no membrane? Here things become more diffi cult
because a protein that carries out a favorable reacti on
(phenotype) will not be physicall y connected to the nucleic
acid (genotype) that encodes it. Thi s consideration suggests
th at th ere mu st be either a "pre-ge nomi c" stage of
evoluti on, where either (1 ) there is a fo rtuitous connection
of protei n and nucleic acid , for exampl e, in a droplet; or
(2) replication and catalysis are carried out by the same
molecules. We often forget that these possibilities are not
mutually exclusive.
* To whom correspondence should be adressed.
Tel: 1-573-882-5668; Fax: 1-573-884-4597
E- mail : schmidtf@ missouri.edu
There is more th an theoreti cal interes t in the first
poss ibility. Recently, complex lipids related to steroids
were fo und in rocks that were 2.7 billi on years old ,
indi cat in g th at bi oc hemi ca ll y di stin c t e uk aryo tes
were likely present earl ier than the appearance of the
oxygen-containing atmosphere (Brocks et ai. , 1999). The
earliest-dated microfossils are about 3.4 bilEon years old
(Schopf and Packer, 1987). Finall y, the abiotic production
of hydrocarbon compounds is well established (Anders
et ai. , 1997, and references therein), so there would have
been a large amount of apolar material available fo r
fo rming such "bubbles."
Regardl ess of whether the first possibility holds, the
natural molecule to carry out dual catalytic and genetic
functi ons is RNA. RNA molecules, being naturally singlestranded, can assume a wide variety of shapes. SELEX
ex perim ents sho w th at virtu all y any li gand , whether
protein (Tuerk and Gold, 1990), nucleic aci d (Cho et ai. ,
1997; Soukop et ai. , 1996), or small molecule (Ellington
and Szostak, 1990; Gold et al., 1995), can be recognized
by a selected, short RNA molecule. Single-stranded DNA
can assume just as many shapes (Sen and Geyer, 1998).
However, since contemporary metabolism derives DNA
precursors from ribonucleotides, we assume the primacy of
an RNA world .
Although contemporary naturall y-occurring ribozymes
act on RNA substrates exclusively, the fact that RNAs can
bind to many small molecules suggests that these RNAs
could have used small molecul e substrates in an RNA
world scenario. Inspired by these res ults, a variety of
laboratories have carri ed out experiments to identi fy
ribozy mes from random librari es. Several themes and
constraints have emerged.
Size of ribozymes Ac ti ve sites of rib ozy mes are
relati vely small . Self-cleaving sequences can be very short,
as in the Mn2+ -dependent ribozyme. First identified by
Hecht and coworkers (Dange et ai., 1990), thi s small
species was reduced to a mere 3 bp by Kazakov and
Altman (1992). The essenti al regions of other species, such
460
Ribozymes -
Why So Many, Wh y So Few?
as the " leadzy me"(Pan and Uhl enbeck, 1994) and the
hammerhead ribozyme (Vaish et ai., 1998), are of simil ar
length . Thi s means that multipl e copi es of acti ve sites
mu st be present in a random library. For example, the
Mn2+ -dependent ribozyme, being onl y 3 bp long, would be
present once every 4096 (46 ) molecules , that is, as often as
an EcoRI restriction site is present in DNA. Since a typical
library used in SELEX has a complexity of 10 14 distinct
species, thi s acti ve site must be present in many molecules.
Thi s effect is illustrated most clearl y by the recent resul ts
of L a nd we be r a nd P o krovs kaya (1999). A library
co nta inin g 101 5 RN As w ith ra nd o m reg io ns 100
nucleotides long was selected for RNA species with guide
RN A- related ligase acti vity. There were two interesting
conclusions. First, onl y 29 nucl eotides were requi red in the
active se lected RNAs, consistent with the idea that acti ve
sites are small. Secondl y, the winnin g molecule also
contained an active site (UUU pai red with GAAA) fo r the
Mn -depe nde nt ribozy me, eve n th o ug h th at was no t
selected for. The fo rtuitous acti ve site was shown to be
catalyticall y acti ve under appropriate conditions. Thi s
result indicates that active sites in ribozymes arise rather
freq uentl y in an RNA of reasonable size, even if they are
not selected fo r. Simpl y put, there are many acti ve sites
availabl e for selecti on to work on.
Trans-acting ribozymes Despite the fac t that ribozyme
ac ti ve sites are relati vely small , the selectio n of any
particul ar ribozyme that acts in trans (and not merely selfcleaves) has generall y requi red the use of a library of long
molecul es. In the first exampl e of such a selection, Szostak
a nd cowo rke rs sy nth es ized a lib ra ry of mo lec ul es
containing 220 random nucleotides (Ekland et ai. , 1995).
More recent selections have empl oyed libraries in the
75- 100 nt range, considerabl y longer than that required for
an acti ve site.
Sa be ti et ai. ( 1997) exa min ed thi s pro bl e m
systemati call y. They calculated that the probabil ity of
ide nti fy ing the hammerhead motif (43 nucleotides) in an
ex pe rim e nt sta rtin g wi th l. 5 X 10 8 molec ul es was
increased 200-fold by increasing the length of the random
sequence 5-fold. This effect is due entirely to the limjted
pool size avail able in an experiment. Obviously if a pool of
443 (7.7 X \025 ) members were avail able for selecti on, the
probability of finding a hammerhead would be 1, since the
libra ry wo uld be ex ha ustiv e ( not a ll bases in th e
hammerhead are unique; the actual probability of finding a
functioning hammerhead is about > 10 10 higher than it
would be if the sequence were unique).
Although it rrught seem that the longer the library the
better, there is a tradeoff operating here. Longer amounts
of ex traneous sequence can interfere with the activity of a
selected RNA. For example, RNase P RNAs can fo ld into
an inacti ve conformation whi ch onl y resolves slow ly into
the optimal structure (Altman, et ai. , 1984). Empiricall y, it
was fo und that a given sequence can inhibit the acti vity of
a li gase ribozyme by nearl y twenty-fold, although other
sequ ences had littl e effect (Sabeti , et ai., 1997). The
conclusion is that longer sequences are better, but onl y for
rare motifs.
Repetitive y ield cons ideration s T he size of th e
molecules available for selecti on has consequences fo r the
ori gin of an RNA world. Any prebiotic or chemical RN A
repli cation or polymerizati on machinery would be poorly
processive, with a low yield per step (Eigen, 1992; Joyce
and Orgel, 1999). A collection of small RNA species made
by such a replicator will necessaril y incl ude many fewe r
"failure sequ ences" th an wo uld a co ll ecti on of longer
polymers. Thi s favors the generation of a coll ection of
small molecul es by any sort of rando m mechani sm or
primitive repli cator. The concentrations, and indeed, the
absolute number, of molecules necessary for selection to
operate would be more easily attained with small rather
than large molecular species. For example, if the repeti tive
yield of a polymerizati on step were 0.9 (actually a rather
good yield by traditional chemi stry), onl y 0.001 % of a total
RNA library would consist of molecules with chain length
~ 100. O ver half the lib ra ry wo ul d be composed of
molecules smaller than 3 residues and 95% composed of
species shorter than 50 res idues. In the example cited
above, the probability of finding a hammerhead would be
reduced - and, by analogy with the arguments above,
much greater than 200-fold. Molecules shorter than the
mjnimal sequence of 43 nucleotides would not be capable
of being selected at all. In other words, ribozymes can onl y
be selected from librari es of lo ng molecul es , bu t the
sy nth e ti c sys te m co uld o nl y ma ke sho rt o nes. Ca n
ribozy mes occur in a pre bi otic world? Or at least a
prebiotic world without a synthesizer and an ample suppl y
of T7 RNA polymerase? We would guess not!
Small RNAs as s ubstrates for ribozyme evolution
In view of thi s consideration, it seems likely that prebiotic
RN A evoluti o n wo uld in volve natural selectio n from
libraries of smaller polymers. The difficulty in identi fy ing
catalysts fro m libraries of short seque nces wo ul d be
addressed by combining two separate small RNA species
to create a primitive ribozy me. In other words, small pi eces
of RNA would have to interact and recombine (ligate) to
make a larger, more efficient ribozyme. We term thi s
process accretion (Fig. 1).
The accreti on model proposes a mechani sm for the
in corporatio n of add itio nal structura l e le me nts into
catalyti c RNAs. We propose that RNA evolution in an
RN A World scenario can occur in a modular fashion by
accretion of di stinct structural elements. Thi s mode could
o pe rate in vitro a nd po te ntia ll y in vivo , a nd wo uld
co mplement the more well-studi ed mechani sm of base
mutagenesis (Beaud ry and Joyce, 1992). Firstl y, initial
Francis 1. Schmidt
GA..,UCUGUAG
cuucuu
U
I I I I I · ..
UCUAG"'CGU~
GAAGAG
111 11 ·
Random RNAs
JGfAU
Itt U
cuuc uu
+
ucJG
11111 ·
GAAGAG
cA Ue
Catalyst
+ Activator
Di ffusion
Transient Active Complex
{
JGrtu
Itt
u
uCUG
Dissociation
~yy~ y~}
GAAGAG
cA Ue
Transesterification
or Ligation
Larger, more active ribozyme
Fig. 1. Accretion model for ribozyme evolution. The model
posits alternate fates for a complex of a ribozyme and its
activator, most often , dissociation and (very) occasionally, the
ligation or transesterification of the complex into a larger species.
ribozymes in a random library would consist of a catalytic
core only. These poorly catalytic, weakly structured RNAs
would be similar to the active sites of contemporary
ribozymes. They can be quite small and would be present
in a collection of small RNAs that arose through random
polymerization of monomers. Secondly, other small,
transactivating RNAs would also be present in the library
of small RNAs. These RNAs would interact transiently
with an existing catalytic species. RNA aptamers selected
to bind an RNA structure bind to their cognate ribozymes
with Ko values in the micromolar, or slightly lower, but
clearly not in the nanomolar, range (Cho et ai., 1997). The
transient nature of these interactions limits their ability to
be selected for. Although it is possible that association
could be mediated by Watson-Crick pairing between
single-stranded regions (Green and Szostak, 1992), it is
unlikely that these regions could be very extensive, as they
are likely to be preferenti a lly degraded in solution.
Furthermore, a paired sequence of 4 bp or less would likely
have a Ko in the micromolar range or greater, and would
th e refore be on ly as stable as the interaction of
transactivating RNAs with the Group I intron (van der
Horst et at., 1991). Next, larger ribozyme variants would
461
ari se throu gh tran sesterification or li gation reaction s
joining small active cores with a transactivating element.
The fact that non-Watson-Crick associations are relatively
weak provides a strong selective force favoring the
incorporation of transactivating domains into a single
polynucleotide chain encompassing multiple domain s.
Finally, association of a catalyst and its activator is
kinetically limited by the relatively weak interaction of the
two RNAs. There is an important co nstraint on the
accretion mechanism. The accretion model posits that
association of two distinct RNA molecules could lead to an
enhancement of catalytic activity and a selecta ble
phenotype. Stable association of the di stinct RNAs to form
larger molecules could be effected by a transesterification
or ligation event, which is known to be RNA-catalyzed
(e.g., Green and Szostak, 1992).
Regardless of whether the tran sesterification catalyst is
intrinsic or extrinsic, there must be a way of getting the
re levant components to interact. However, non-WatsonCrick RNA-RNA interaction s are relatively weak with
Ko 's in the range of 0. 1 to 2 /J.M. Thi s is too small for a
stable association. If the diffusion-limited association rate
is 108 M - 1S - I, then the di ssociation rate constant, koff ' of
the complex is given by:
Thi s rate can be compared to the rate of the chemical
step of tran sesterification (Herschlag and Cech , 1990)
which is 350 min - I, or 6 s - I . Thus, di ssociation of any
non-Watson-Crick complex between two small RNAs is at
least an order of magnitude faster than a potential RNAcatalyzed event that could join them together. At most,
then, only 1- 10% of all encounters would be capable of
being joined into a covalent array even if the original
ribozyme catalyzed some sort of tran sesterification or
ligation event. If the joining event occurred through the
intervention of another catalyst, then the rate of reaction
would be even lower, and dependent on the concentration
of the catalyst.
One might think that these considerations preclude the
association of two RNAs in a random popul ation from ever
interacting in a finite period of time, and , indeed, we agree
that thi s wou ld preclude any two specific molecules from
ever corning together. The accretion model , however,
posits that many molecules in a population share the ability
to transactivate a given ribozyme. If, for example, 10% of
all the small RNAs were capable of transactivation , then
the effective concentration of transactivating RNAs would
be 0.1 X the total [RNA] in the library. If only a single
mo lecu Ie were able to tran sac ti vate, the effecti ve
concentration would be 10- 12 X the total [RNA] . Thi s
consideration relates to the probability of an RNA being
transesterified or ligated into a larger molecule. If the
proportion of activating molecu les is 10- 6 , then the
Ribozy mes -
462
Why So Many, Why So Few?
effective KD of the noncovalent complex that is capable of
transactivation would be increased by 106 , that is, it wou ld
essentiall y be unity, and the effective koff would be
correspondingly increased to 10 8 s - I. "Fixation" of a
transactivating RNA could potentially occur in only one
encounter of 107_10 8. On the other hand, if there were only
a single transactivator in the population of 421 mol ecules,
the effective di ssociation constant would be 106 . In thi s
14
case th e effective korf would be 10 S - I, and s uch
" fixation " cou ld lead to a novel RNA on ly one time
14
in 10 .
The effectiveness of fixation is limited by the rate
of deg rad at ion of the interactin g RNA mol ec ul es,
11
effectively 5 X 10- per phosphodiester bond per second
(Hersch lag and Cech , 1990). If we assume for the sake of
argument that there are 10 crucial phosphodiester bonds
per catalytic RNA, then the half-life of such a molecul e
wou ld be approximately
In2
10 * k
9
2.4 X 10 s
If on ly one molecule in the random population were
ab le to tr a nsactivate, the expected numb er of
transactivations per catalyst during its lifetime would be
less than 3 X 10- 4 . On the other hand, if transactivating
molecules occurred at a frequency of 10- 6 in a random
collection of species, then the effective rate of di ssociation
would be 10 8 s - I. In its lifetime, therefore, a molecule
would likely be tran sac tivated > 10 times before it
degraded . If the accretion model is to be viable, we assume
that an RNA should encounter a transactivator at least once
during its lifetime. In other words, the effective rate of
association must be roughl y 10- 7 s - I .
RNA interactions The possibility of these interactions is
hinted at by the structure of contemporary large ribozymes,
whose arc hitecture involves non-Watson-Crick interaction
between preformed dom ains. The Group I intron from
Tetrahymena forms a comp lex between two domains, with
the ac tive site at the junction between them (Golden et al. ,
1998 ). More to the point is the ph e nomenon of
tran sacti vation . Small structural elements can be deleted
from both the Group I intron (van der Horst, 1991 ) and
RN ase P RNA (Kim and Schmidt , unpubli s h e d
observations) and then added back. Deletion of any single
structural e le me nt does not aboli s h catalytic activity
although it dimini s hes it co nsiderably by making the
ribozyme structure less stable (Darr et al., 1992). Addition
of the de leted structure in trans restores catalytic activity,
although not to wi ld-type levels. Thi s means that there is
a selective pressure to increase the size of the ribozyme
beyond the size of the active site elements alone, and
provides an evolutionary argument for the large size of
contemporary biocatalysts (Narliker and Herschl ag, 1998).
Acknowledgements I th ank Dr. Hado ng Kim, Ri c hard
Poelling, Tom Leeper, Melissa Meyer and David Taylor for their
enthusiastic participati on in thi s work, which was supported by
the Un iversity of Missouri Research Board .
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