Ribozymes

Altman and Cech Nobel Prize in Chemistry 1989

The long held belief that enzymes could only be proteins has come to an end. In 1989 investigators Sidney Altman and Thomas R. Cech were awarded the Nobel Prize in Chemistry for their discovery of catalytic properties of RNA, unmasking RNA's potential as a totally new type of enzyme. These RNA enzymes, called ribozymes, have posed an interesting new set of challenges for researchers in determining the exact structure and function of these molecules. Ribozymes have also provided a new shot of inspiration to all of humanity as the possibilities for using them in fighting viral infections and in explaining the very existence of life itself have come to light.

Several new examples of these ribozymes are currently under investigation. In order for a RNA molecule to qualify as an enzyme it must act as a catalyst in biochemical reactions. Catalysts are molecules affecting the rate of a chemical reaction without being used up or affected themselves. Enzymes are also defined as being highly specific in their catalytic functions. Strands of circular ribozymes, called viroids, have been discovered which satisfy these conditions and can have a devastating effect on plants. The ribozymes replicate themselves in copies attached to their own genome. The viroids then undergo self-cleavage, sending fragments off to colonize other areas of the plant. The viroids harm the plants by rapidly proliferating and using nucleotide materials the plant itself needs. Further damage is caused as the viroid bundles interfere with the plant’s internal structures much like a tumor. They have no protein producing capabilities. Their cleavage from the mother strand is completely self-controlled and initiated. They are catalytic in their own replication and processing.

Ribozymes are RNA Enzymes

Researchers have isolated a specific site in these viroids responsible for the process of self-cleavage. The site is less than 30 nucleotides long and consists of three stems coming off a central loop. This secondary structure, called a "hammerhead", is capable of cleaving very specific sequences of RNA in order to release viable daughter strands of RNA. Synthetic hammerheads, consisting of only 19 nucleotides, have already been produced, which can act as highly specific catalysts. Similar synthetic ribozymes are being designed to break up RNA viruses and RNA involved in the transcription and translation of mutant DNA.

Another significant ribozyme is ribonuclease P (RNase P). RNase P is able to selectively cut more than 60 tRNA precursors, which then become mature tRNA molecules capable of carrying amino acids during the translation of proteins. Without RNase P this translation process would not be possible. The enzyme is a ribonucleoprotein, although the RNA segment of the molecule has been shown to independently recognize and cleave the appropriate substrate both in vivo and in vitro. The protein segment of the RNase P appears to allow the ribozymal segment to work at a faster hydrolytic rate and with less Mg2+ present. Ribozymes often appear to bridge the gap between DNA and proteins. This observation, combined with the presence of the ribonuclease in all living organisms, indicates a long history of involvement with the development of life.

One of the best understood of the ribozymes is L19 RNA from the protozoan, Tetrahymena. L19 RNA is involved in the processing of rRNA. The ribozyme begins as an intron, or noncoding region, of RNA. The first task of L19 RNA is to cleave itself from the coding exons of the RNA precursor. Guanosine or a guanine nucleotide (GMP, GDP, or GTP) attaches to the 5’ end of the intron forming a phosphodiester bond. This bond formation produces a 3’-OH at the end of the exon causing it to separate from the intron. This 3’-OH then attacks the 3’ end of the intron still attached to the other half of the exon. The exon halves join as the intron is pinched out from the molecule. At this point the ribozymal intron is a linear strand of RNA composed of 414 base pairs with a guanosine unit attached to the 5’ end and a 3’-OH tail. The 3’-OH end then bonds just downstream from its own 5’ end. The resulting form looks like a lasso until the linear portion of 19 base pairs breaks away leaving only the loop portion. This loop opens and closes again in the same pattern as before, this time cutting off 4 base pairs. Again the loop opens, forming a linear strand of 395 nucleotides called L19 RNA.

L19 Tetrahymena RNA ribozyme      mechanism. Keep pressing the "Next"      arrow to view the animation.

L19 RNA is not only able to cleave itself, but is able to both lengthen and shorten the size of the oligoribonucleotides such as pentacytidylate (C5) in rRNA. Oligoribonucleotide is the name given to a short segment of RNA. This molecule first forms hydrogen bonds with the G rich 5’ end of the ribozyme. Then a G from the 3’ end of L19 RNA breaks the phosphodiester bond of the oligoribonucleotide and strips off the last C releasing C4. The ribozyme can either release its captive C through hydrolysis or combine with another C5 where it will actually add on the C forming a C6. L19 RNA, in effect, converts C5 present into C4, C3, and C6 or higher.

Oligoribonucleotide shortening by L19 RNA.

Because of their enzymatic properties, ribozymes are today considered by many to be the first evolutionary step towards life. Their small size makes them relatively simple molecules. This means there is a much higher possibility for them to occur spontaneously in a prehistoric UV-baked pool filled with nucleotide precursors. They possess the ability to both replicate themselves and to manipulate proteins. Ribozymes have been shown to catalyze a wide variety of chemical reactions including isomerizations, hydrolytic reactions, phosphoryl group transfers and many other extremely important interactions, both in terms of energy storage, energy utilization and for both catabolic and anabolic processes. They are essentially gifted with the talents of both DNA and protein enzymes. Mutations and selective pressures may have led to the proliferation of those ribozymes that manipulated molecules to store their heritable information or complete their enzymatic chores for them. The viroids currently under examination may possess the greatest resemblance to the first ancestor of all life.

Due to their simplicity and multi-task functions, the possibilities for ribozymes in the scientific community are many. A ribozyme has already been approved for testing in HIV patients. The ribozyme has been made to attack and break up the RNA composing the virus itself. Ribozymes may also be used in the future to correct genetic disorders. Unhealthy proteins may be eliminated, essentially before they exist, by breaking up the RNA molecules responsible for translating and transcribing them.