3D structure of a hammerhead ribozyme
Ribozymes (ribonucleic acid enzymes) are RNA molecules that are capable of catalyzing specific biochemical reactions, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonstrated that RNA can be both genetic material (like DNA) and a biological catalyst (like protein enzymes), and contributed to the RNA world hypothesis, which suggests that RNA may have been important in the evolution of prebiotic self-replicating systems.
The most common activities of natural or in vitro-evolved ribozymes are
the cleavage or ligation of RNA and DNA and peptide bond formation. Within the ribosome, ribozymes function as part of the large subunit ribosomal RNA to link amino acids during protein synthesis. They also participate in a variety of RNA processing reactions, including RNA splicing, viral replication, and transfer RNA biosynthesis. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme, Leadzyme and the hairpin ribozyme.
Investigators studying the origin of life have produced ribozymes in the laboratory that are capable of catalyzing their own synthesis from activated monomers under very specific conditions, such as an RNA polymerase ribozyme. Mutagenesis
and selection has been performed resulting in isolation of improved
variants of the "Round-18" polymerase ribozyme from 2001. "B6.61" is
able to add up to 20 nucleotides to a primer template in 24 hours, until it decomposes by cleavage of its phosphodiester bonds. The "tC19Z" ribozyme can add up to 95 nucleotides with a fidelity of 0.0083 mutations/nucleotide.
Attempts have been made to develop ribozymes as therapeutic
agents, as enzymes which target defined RNA sequences for cleavage, as biosensors, and for applications in functional genomics and gene discovery.
Discovery
Schematic showing ribozyme cleavage of RNA.
Before the discovery of ribozymes, enzymes, which are defined as catalytic proteins, were the only known biological catalysts. In 1967, Carl Woese, Francis Crick, and Leslie Orgel were the first to suggest that RNA could act as a catalyst. This idea was based upon the discovery that RNA can form complex secondary structures. These ribozymes were found in the intron
of an RNA transcript, which removed itself from the transcript, as well
as in the RNA component of the RNase P complex, which is involved in
the maturation of pre-tRNAs. In 1989, Thomas R. Cech and Sidney Altman shared the Nobel Prize in chemistry for their "discovery of catalytic properties of RNA." The term ribozyme was first introduced by Kelly Kruger et al. in 1982 in a paper published in Cell.
It had been a firmly established belief in biology
that catalysis was reserved for proteins. However, the idea of RNA
catalysis is motivated in part by the old question regarding the origin
of life: Which comes first, enzymes that do the work of the cell or
nucleic acids that carry the information required to produce the
enzymes? The concept of "ribonucleic acids as catalysts" circumvents
this problem. RNA, in essence, can be both the chicken and the egg.
In the 1980s Thomas Cech, at the University of Colorado at Boulder, was studying the excision of introns in a ribosomal RNA gene in Tetrahymena thermophila.
While trying to purify the enzyme responsible for the splicing
reaction, he found that the intron could be spliced out in the absence
of any added cell extract. As much as they tried, Cech and his
colleagues could not identify any protein associated with the splicing
reaction. After much work, Cech proposed that the intron sequence
portion of the RNA could break and reform phosphodiester bonds. At about the same time, Sidney Altman, a professor at Yale University,
was studying the way tRNA molecules are processed in the cell when he
and his colleagues isolated an enzyme called RNase-P, which is
responsible for conversion of a precursor tRNA
into the active tRNA. Much to their surprise, they found that RNase-P
contained RNA in addition to protein and that RNA was an essential
component of the active enzyme. This was such a foreign idea that they
had difficulty publishing their findings. The following year, Altman
demonstrated that RNA can act as a catalyst by showing that the RNase-P
RNA subunit could catalyze the cleavage of precursor tRNA into active
tRNA in the absence of any protein component.
Since Cech's and Altman's discovery, other investigators have
discovered other examples of self-cleaving RNA or catalytic RNA
molecules. Many ribozymes have either a hairpin – or hammerhead – shaped
active center and a unique secondary structure that allows them to
cleave other RNA molecules at specific sequences. It is now possible to
make ribozymes that will specifically cleave any RNA molecule. These RNA
catalysts may have pharmaceutical applications. For example, a ribozyme
has been designed to cleave the RNA of HIV. If such a ribozyme were
made by a cell, all incoming virus particles would have their RNA genome
cleaved by the ribozyme, which would prevent infection.
Structure and mechanism
Despite
having only four choices for each monomer unit (nucleotides), compared
to 20 amino acid side chains found in proteins, ribozymes have diverse
structures and mechanisms. In many cases they are able to mimic the
mechanism used by their protein counterparts. For example, in self
cleaving ribozyme RNAs, an in-line SN2 reaction is carried out using the
2’ hydroxyl group as a nucleophile attacking the bridging phosphate and
causing 5’ oxygen of the N+1 base to act as a leaving group . In
comparison, RNase A, a protein that catalyzes the same reaction, uses a
coordinating histidine and lysine to act as a base to attack the
phosphate backbone.
Like many protein enzymes metal binding is also critical to the function of many ribozymes.
Often these interactions use both the phosphate backbone and the base
of the nucleotide, causing drastic conformational changes.
There are two mechanism classes for the cleavage of phosphodiester
backbone in the presence of metal. In the first mechanism, the internal
2’- OH group attacks phosphorus center in a SN2 mechanism.
Metal ions promote this reaction by first coordinating the phosphate
oxygen and later stabling the oxyanion. The second mechanism also
follows a SN2 displacement, but the nucleophile comes from
water or exogenous hydroxyl groups rather than RNA itself. The smallest
ribozyme is UUU, which can promote the cleavage between G and A of the
GAAA tetranucleotide via the first mechanism in the presence of Mn2+.
The reason why this trinucleotide rather than the complementary
tetramer catalyze this reaction may be because the UUU-AAA pairing is
the weakest and most flexible trinucleotides among the 64 conformations,
which provides the binding site for Mn2+.
Phosphoryl transfer can also be catalyzed without metal ions. For
example, pancreatic ribonuclease A and hepatitis delta virus(HDV)
ribozymes can catalyze the cleavage of RNA backbone through acid-base
catalysis without metal ions. Hairpin ribozyme can also catalyze the self-cleavage of RNA without metal ions but the mechanism is still unclear.
Ribozyme can also catalyze the formation of peptide bond between adjacent amino acid by lowing the activation entropy.
Image showing the diversity of ribozyme structures. From left to right: leadzyme, hammerhead ribozyme, twister ribozyme
Activity
Although most ribozymes are quite rare in the cell, their roles are
sometimes essential to life. For example, the functional part of the ribosome, the molecular machine that translates RNA into proteins, is fundamentally a ribozyme, composed of RNA tertiary structural motifs that are often coordinated to metal ions such as Mg2+ as cofactors. In a model system, there is no requirement for divalent cations in a five-nucleotide RNA catalyzing trans-phenylalanation
of a four-nucleotide substrate with 3 base pairs complementary with the
catalyst, where the catalyst/substrate were devised by truncation of
the C3 ribozyme. RNA may catalyze folding of the pathological protein conformation of a prion in a manner similar to that of a chaperonin, and may be involved in the viral concatemer cleavage that precedes the packing of viral genetic material into some virons.
RNA can also act as a hereditary molecule, which encouraged Walter Gilbert to propose that in the distant past, the cell used RNA as both the genetic material and the structural and catalytic molecule rather than dividing these functions between DNA and protein as they are today; this hypothesis is known as the "RNA world hypothesis" of the origin of life.
Evidence that ribozymes were the first molecular machines used by early
life suggests that they are in effect "molecular fossils".
Artificial ribozymes
Since
the discovery of ribozymes that exist in living organisms, there has
been interest in the study of new synthetic ribozymes made in the
laboratory. For example, artificially-produced self-cleaving RNAs that
have good enzymatic activity have been produced. Tang and Breaker
isolated self-cleaving RNAs by in vitro selection of RNAs originating
from random-sequence RNAs. Some of the synthetic ribozymes that were
produced had novel structures, while some were similar to the naturally
occurring hammerhead ribozyme. In 2015, researchers at Northwestern University and the University of Illinois
at Chicago have engineered a tethered ribosome that works nearly as
well as the authentic cellular component that produces all the proteins
and enzymes within the cell. Called Ribosome-T, or Ribo-T, the artificial ribosome was created by Michael Jewett and Alexander Mankin.
The techniques used to create artificial ribozymes involve directed
evolution. This approach takes advantage of RNA's dual nature as both a
catalyst and an informational polymer, making it easy for an
investigator to produce vast populations of RNA catalysts using polymerase enzymes. The ribozymes are mutated by reverse transcribing them with reverse transcriptase into various cDNA and amplified with error-prone PCR. The selection parameters in these experiments often differ. One approach for selecting a ligase ribozyme involves using biotin tags, which are covalently linked to the substrate. If a molecule possesses the desired ligase activity, a streptavidin matrix can be used to recover the active molecules.
Lincoln and Joyce developed an RNA enzyme system capable of self
replication in about an hour. By utilizing molecular competition (in vitro
evolution) of a candidate RNAmixture, a pair of ribozymes emerged, in
which each synthesizes the other by joining synthetic oligonucleotides,
with no protein present.
Although not true catalysts, the creation of artificial
self-cleaving riboswitches, termed aptazymes, has also been an active
area of research. Riboswitches
are regulatory RNA motifs that change their structure in response to a
small molecule ligand to regulate translation. While there are many
known natural riboswitches that bind a wide array of metabolites and
other small organic molecules, only one ribozyme based on a riboswitch
has been described, glmS. Early work in characterizing self-cleaving riboswitches was focused on using theophylline as the ligand. In these studies an RNA hairpin is formed which blocks the ribosome binding site, thus inhibiting translation. In the presence of the ligand,
in these cases theophylline, the regulatory RNA region is cleaved off,
allowing the ribosome to bind and translate the target gene. Much of
this RNA engineering work was based on rational design and previously
determined RNA structures rather than directed evolution as in the above
examples. More recent work has broadened the ligands used in ribozyme
riboswitches to include thymine pyrophosphate (2).
Fluorescence-activated_cell_sorting has also been used to engineering
aptazymes.
Applications
Ribozymes
have been proposed and developed for the treatment of disease through
gene therapy (3). One major challenge of using RNA based enzymes as a
therapeutic is the short half-life of the catalytic RNA molecules in the
body. To combat this, the 2’ position on the ribose is modified to
improve RNA stability. One area of ribozyme gene therapy has been the
inhibition of RNA-based viruses.
A type of synthetic ribozyme directed against HIV RNA called gene shears has been developed and has entered clinical testing for HIV infection.
Similarly, ribozymes have been designed to target the hepatitis C virus RNA and influenza A and B virus RNA.
The ribozyme is able to cleave the conserved regions of the virus’s
genome which has been shown to reduce the virus in mammalian cell
culture. Despite these efforts by researchers, these projects have remained in the preclinical stage.
