Page 88 - Academic Press Encyclopedia of Physical Science and Technology 3rd Molecular Biology

P1: GRB/GRD P2: GNB Final Pages
Encyclopedia of Physical Science and Technology EN014F-661 July 28, 2001 20:35

260 Ribozymes

Generalized Hairpin Ribozyme

Cleavage site
Cleavage site
3í 3í
H2
H1 U U H2 5í 5í
H1
C C G G N N
YRNN
NNNNNN YRNN
NNNNNN
| | | | | | | | |
| | | | | | | | | | |
GYNNN--NN
NNNNNN GYNNN--NN 3í 3í
NNNNNN
N--N
N N A A A A N--N
G G N--N
N--N
N--N H3
N--N
H3
N--N
5í N--N
N N C C
A A
G G U U
A A U U
A A A A N N A A
N
A A N
C N
C N
N N
N--N
N--N
H4
N--N H4
N--N
N--N
N--N
N N N N
N N
FIGURE 3 Generalized hairpin ribozyme. The target RNA (upper sequence) base pairs with the ribozyme (lower
sequence) by Watson–Crick base-pairing. Cleavage occurs at the site indicated. The helices formed by the ribozyme
and substrate are designated as H1–H4, as indicated. N = any nucleotide, Y = pyrimidine, P = purine. Other bases
are A = adenosine, C = cytosine, G = guanosine, and U = uracil.
mutant lacZ transcript, first in bacteria and subsequently sequenced, and the complement to the ribozyme pairing
in correction of a sickle cell message in erythroid cells. arms was determined. The identity of the target revealed
a novel tumor suppressor. This approach has potential
applications in many systems (therapeutic or otherwise)
G. Ribozyme Based Gene Discovery where a phenotype is of interest but the genes involved are
unknown.
The necessity of ribozymes to selectively base-pair with
the target RNAs prior to catalysis has been exploited to
discover new gene functions involved in a particular phe- H. Ribozyme Evolution
notype.Thisprotocolhasbeentermed“inversegenomics.”
Acombinatorialribozymelibraryhasbeenintroducedinto The discovery of the ribozyme sparked new debate on the
cultured cells, and selection or scoring for a particular phe- “RNA world” hypothesis, where all biological processes
notype has led to the identification of several new gene were carried out by RNA-based enzymes. Since then,
functions. A hairpin ribozyme library containing roughly RNA evolution has been forced in vitro to come up with
7
2 × 10 different ribozymes was stably transduced into RNA enzymes capable of carrying out a wide variety of
tumor cells in culture and cells able to grow in soft agar biochemical reactions, as far-reaching as carbon carbon
were identified. Ribozymes isolated from these cells were bond and peptide bond formation. In vitro RNA evolution