Structure of hammerhead ribozyme

A ribozyme is an RNA molecule with a well defined tertiary structure that enables it to perform a chemical reaction. Many ribozymes are catalytic, but some such as self-cleaving ribozymes are consumed by their reactions. Ribozyme means ribonucleic acid enzyme. It may also be called an RNA enzyme or catalytic RNA. It contains an active site that consists entirely of RNA. Many natural ribozymes cleave one of their own phosphodiester bonds (self-cleaving ribozymes), or bonds in other RNAs. Some have been found to catalyze the aminotransferase activity of the ribosome. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme and the hairpin ribozyme.

Investigators studying the origin of life have produced ribozymes in the laboratory that are capable of catalyzing their own synthesis under very specific conditions, such as an RNA polymerase ribozyme.^[1] 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.^[2] The "tC19Z" ribozyme can add up to 95 nucleotides with a fidelity of 0.0083 mutations/nucleotide.^[3]

Some ribozymes may play an important role as therapeutic agents, as enzymes which tailor defined RNA sequences, as biosensors, and for applications in functional genomics and gene discovery.^[4]

Discovery[link]

Schematic showing ribozyme cleavage of RNA.

Before the discovery of ribozymes, enzymes, which are defined as catalytic proteins,^[5] 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.^[6] The first ribozymes were discovered in the 1980s by Thomas R. Cech, who was studying RNA splicing in the ciliated protozoan Tetrahymena thermophila and Sidney Altman, who was working on the bacterial RNase P complex. 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 won the Nobel Prize in chemistry for their "discovery of catalytic properties of RNA."^[7] The term ribozyme was first introduced by Kelly Kruger et al. in 1982 in a paper published in Cell.^[8]

It had been a firmly established belief in biology that catalysis was reserved for proteins. In retrospect, catalytic RNA makes a lot of sense. This is based on 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? Ribo-Nucleic acids as catalysts circumvents this problem. RNA, in essence can be both the chicken and the egg.^[9]

In the 1970s 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 splicing reaction, he found that 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 was made by a cell, all incoming virus particles would have their RNA genome cleaved by the ribozyme, which would prevent infection.

Activity[link]

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. There is no requirement for divalent cations in a five-nucleotide RNA that can catalyze trans-phenylalanation of a four-nucleotide substrate which has three base complementary sequence with the catalyst. The catalyst and substrate were devised by truncation of the C3 ribozyme.^[10]

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 became known as the "RNA world hypothesis" of the origin of life.

If ribozymes were the first molecular machines used by early life, then today's remaining ribozymes—such as the ribosome machinery—could be considered living fossils of a life based primarily on nucleic acids.

A recent test-tube study of prion folding suggests that an RNA may catalyze the pathological protein conformation in the manner of a chaperone enzyme.^[11]

Ribozymes have been shown to be involved in the viral concatemer cleavage that precedes the packing of viral genetic material into virions.^[12][13]

Known ribozymes[link]

Naturally occurring ribozymes include:

Artificial ribozymes[link]

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^[15] 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.

The techniques used to discover artificial ribozymes involve Darwinian 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 mutagenic 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 enzyme mixture, a pair of RNA enzymes emerged, in which each synthesizes the other from synthetic oligonucleotides, with no protein present.^[16]

Applications[link]

A type of synthetic ribozyme directed against HIV RNA called gene shears has been developed and has entered clinical testing for HIV infection.^[17][18]

See also[link]

References[link]

Further reading[link]

External links[link]

• Thomas Cech iBioMagazine talk: "Discovering Ribozymes"