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Encyclopedia of Physical Science and Technology EN014F-661 July 28, 2001 20:35

Ribozymes 255

catalyze reactions on exogenous substrates. The ability of stranded RNA substrate composed of two separate RNA
the group II introns to bind the 5 -exon specifically has molecules was also a suitable cleavage substrate. Mixing

been exploited to encourage the IVS to catalyze reactions two short complementary oligoribonucleotides containing

on exogenous substrates. These introns can be engineered a3 -proximal NCCA sequence resulted in cleavage of the
to insert into target RNAs in trans in a reversal of the splic- target RNA at the predicted site. It seems, then, that any
ing reaction, thereby making them useful for site-specific RNA can be cleaved by endogenous RNase P if an exter-
gene inactivation or site-specific integration of therapeutic nal guide sequence (EGS) containing a single-stranded
genes. NCCA at its 3 -end is provided to hybridize with the

chosen target.
III. RIBONUCLEASE P
IV. SELF-CLEAVING RNAS
Ribonuclease P (RNase P) is a ubiquitous endoribonucle-

asethatprocessesthe5 -endofprecursortRNAmolecules, One category of intramolecular RNA catalysis is that

producing a 5 -phosphate and 3 -OH termini on the cleav- which produces a 2 ,3 -cyclic phosphate and 5 -OH ter-

age products. RNase P consists of both protein and RNA minus on the reaction products. A number of small plant
components, and it was shown that the catalyst was pathogenic RNAs (viroids, satellite RNAs, and virusoids),
the RNA moiety. As with the catalytic introns, a diva- a transcript from a Neurospora mitochondrial DNA plas-
lent cation is required as cofactor. RNase P is unique mid, and the animal HDV undergo a self-cleavage reaction
among naturally occurring ribozymes in that it binds and in vitro in the absence of protein. The reactions require
2+
cleaves free substrate molecules; all other characterized neutral pH and Mg . It is thought that the self-cleavage
ribozymes act in cis. This natural trans-activity makes reaction is an integral part of their in vivo rolling circle
RNase P an obvious candidate for development as a ther- mechanism of replication. These self-cleaving RNAs can
apeutic agent. Another feature that distinguishes RNase be subdivided into groups depending on the sequence and
P from all other ribozymes is that it does not involve secondary structure formed around the cleavage site.
Watson–Crick base-pairing between the catalytic RNA
and substrate for substrate recognition. Much effort there-
A. Hammerhead Ribozymes
fore has been directed toward elucidating the biochemistry
and substrate specificity of the RNase P cleavage reaction. This group of RNAs shares a two-dimensional struc-
One of the first aspects to be analyzed was the role of tural motif known as the hammerhead, which has been
the protein subunit in the cleavage reaction. Comparisons shown to be sufficient to direct site-specific cleavage. The
of the kinetic aspects of the B. subtilis RNA-dependent hammerhead structure consists of three base-paired stems
cleavage reaction performed under various ionic condi- which flank the susceptible phosphodiester bond, and two
tions have demonstrated that high ionic strength and ad- single-stranded regions, which are highly conserved in se-
dition of the protein subunit have similar effects on the quence. Extensive mutagenesis has revealed the important
kinetics of cleavage. This may indicate that the protein nucleotides and functional groups for efficient catalysis.
subunit acts to disperse the charge repulsions between the The hammerhead cleavage domain has been split into two
RNase P and precursor tRNA substrate RNAs. Because or three independent RNAs, and trans-cleavage has been
many RNase P RNAs are not functional in the absence demonstrated in vitro. Haseloff and Gerlach proposed a
of protein in vitro, another possible role for the protein model whereby the hammerhead domain is separated such
is to help the RNA moiety to fold into the proper confor- that the substrate RNA contains just the cleavage site, and
mation. As with most RNA-processing enzymes, the exact the ribozyme contains the other conserved nucleotides of
substraterequirementsintermsofsequenceandsecondary the catalytic core. Mutagenesis has revealed that the target
structure are not well understood. It has been shown that site can be any NUH sequence where H = A, C, and N is
mature tRNA can compete for binding, suggesting that any nucleotide. The sequence of the arms of the ribozyme
most of the binding energy comes from mature tRNA. aligns the catalytic core to the target site via complemen-
Analysis of pre-tRNA deletion mutants showed that only tary base-pairing, Analysis has also allowed determination
the amino acceptor stem and the T loop and stem, which of the minimum core sequence required for catalytic ac-
form a single coaxially stacked helix, are required for tivity. The ability to cleave the RNA and thereby inhibit
cleavage by RNase P, suggesting that any hairpin struc- the expression of a specific gene selectively has two main
ture can be cleaved by RNase P provided that a single- applications: as a surrogate genetic tool for molecular bi-

stranded NCCA trinucleotide is present at the 3 -side of ology and the inactivation of gene transcripts in vivo,as
the hairpin. Forster and Altman asked whether a double- antiviral agents, for example.