Ribozyme Catalysis and Structure
Ribozymes are antisense RNA molecules that have catalytic activity. They
function by binding to the target RNA moiety through Watson-Crick base
pairing and inactivate it by cleaving the phosphodiester backbone at a
specific cutting site.
Five classes of ribozymes have been described based on their unique
characters in the sequences as well as three-dimensional structures
(Bunnell,1997). They are denoted as (1) the Tetrahymena group I intron, (2)
RNase P, (3) the hammerhead ribozyme, (4) the hairpin ribozyme, and (5)
the hepatitis delta virus ribozyme. They may catalyze self-cleavage
(intramolecular or "in-cis" catalysis) as well as the cleavage of external
substrates (intermolecular or "in-trans" catalysis) (Ohkawa, 1995). The
leading study was conducted by Thomas Cech on ciliated protozoan
Tetrahymena Thermophila group I introns in 1980s, which showed that RNA
can participate in the intramolecular catalysis of the self-splicing and acts
as a protein enzyme. The definition of biological enzyme was broadened
since then, by recognizing the enzymatic function of some RNA molecules.
Figure 1. Tetrahymena Group I intron secondary and tertiary structures
(Golden B., Cech T.)
P4-P6 Structure (Doudna J. , Cech T.)
Figure 2. A hammerhead ribozyme structure with magnesium cation
binding sites (Murray,1996)
The name of hammerhead ribozyme is given by the similarity between its
secondary structure and the shape of a hammerhead. They are the best
understood subcategory of all ribozymes. As well as other ribozymes, the
hammerhead ribozyme is an antisense RNA. Some of the ribonucleotides
within the sequence selectively form Watson-Crick base pairs with others to
form a stem, while the rest stay in single stranded state called loop. These
loops and stems can be predicted at the secondary structure level using
conformational energy analysis, such as RNAdraw and mfold; and three
dimensional structures were obtained mainly by X-ray crystallography.
Figure 3. Secondary structures of in cis and in trans hammerhead
ribozymes
On the left of Figure 3, the structure of the wild-type, cis-acting
hammerhead ribozyme is shown. The three helices and the cleavage site are
indicated. The self-splicing happens after the sequence G-U-C between helix
I and III, and the G and U are conserved and crucial for the catalysis. Except
for itself, a hammerhead ribozyme is not catalytic in cells since its nature of
intramolecular reaction (Symons, 1989). However, most ribozymes can be
chemically engineered to cleave RNA in trans by separating the catalytic
domain from ribozyme and attaching recognition (i.e., antisense) arms to
the catalytic center, in order to target to a substrate. These trans-acting
ribozymes can be very useful in the study of molecular biology and
pharmaceutics.
In comparison to the conventional antisense RNAs, ribozymes provide
the potential of turnover, with a single molecule being able to inactivate
multiple target RNAs. Another important property of ribozyme is the
specificity,
Figure 4. Turnover cycle of RNA cleavage by a hammerhead ribozyme.
Binding of the enzyme and substrate results in the catalytically active
structure. After cleavage, the two product strands dissociate and the
ribozyme strand can go into the next cycle.
which means the fidelity to cleave a unique target. Both of turnover and
specificity are affected by binding arm length (helix I and III). If the length
of binding arms is very short, the rate of dissociation of the target from the
ribozyme may exceed the rate of cleavage, resulting in poor efficiency
(Rossi, 1997). However, stable hybrids exhibit low catalytic activity because
of slow dissociation of the cleaved substrate (Bertrand, 1994). Thus, the
ideal situation is to have arm lengths that aid cleavage, yet provide for
quick dissociation of the cleaved products.
This present web page is part of a term paper assignment for the Advanced
Enzymology Class (Quan Du).