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Chapter: Biotechnology Applying the Genetic Revolution: RNA-Based Technologies

Small Naturally Occurring Ribozymes

In contrast to the large ribozymes, small ribozymes are only about 200 to 500 nucleotides long. Small ribozymes include the hammerhead and hairpin ribozymes, the hepatitis delta virus ribozyme, and the Varkud satellite ribozyme.

SMALL NATURALLY OCCURRING RIBOZYMES

In contrast to the large ribozymes, small ribozymes are only about 200 to 500 nucleotides long. Small ribozymes include the hammerhead and hairpin ribozymes, the hepatitis delta virus ribozyme, and the Varkud satellite ribozyme. These are often found in viroids, virusoids, and satellite viruses, which are subviral agents. Viroids are self-replicating pathogens of plants that are merely naked single strands of RNA with no protein coat. Satellite viruses are small RNA molecules that require a helper virus for either replication or capsid formation. Their genomes may encode proteins. Virusoids are even less functional and are often considered a subtype of satellite virus. Virusoids are single strands of circular RNA that encode no proteins. They rely on helper viruses for both replication and a protein coat.

 

The hammerhead ribozyme is a small catalytic RNA that can catalyze a self-cleavage reaction. Hammerhead ribozymes take part in the replication of some viroids and satellite RNAs (Fig. 5.23). These both exist as single-stranded RNA genomes that form rodlike structures that are resistant to cellular ribonucleases. During viroid replication, the positive RNA strand is replicated by the host cell RNA polymerase, resulting in a long concatemer of negative-strand genomes. RNA polymerase then uses this as template to make a positive strand. The long RNA is cut into individual unit genomes by the hammerhead motif. Hammerhead ribozymes first cleave the ribose phosphate backbone of RNA, and then ligate the linear unit genomes into circular genomes.




Another small ribozyme is the hairpin ribozyme (Fig. 5.24). This is found in pathogenic plant satellite viruses such as tobacco ring spot virus. The hairpin ribozyme from tobacco ring spot virus was originally called the “paperclip” ribozyme, and this may be a better description of the structure.

 

In vivo, hairpin ribozymes cleave the linear concatemers of single-stranded RNA genomes, much like hammerhead ribozymes, and then ligate the linear segments into circular genomes.

 

Two other small ribozymes are the Varkud satellite ribozyme from Neurospora and the hepatitis delta virus (HDV) of humans. Both work by similar reaction mechanisms to initiate self-cleavage and ligation reactions.



The Varkud satellite ribozyme helps replicate the small Varkud plasmid found within the mitochondria of  Neurospora. The hepatitis delta virus is a viroid-like satellite virus of the hepatitis B virus.

Hepatitis B infects the liver and can cause liver scarring, liver failure, and sometimes death.

 

In patients with hepatitis B, the presence of HDV amplifies the symptoms, causing a very severe and often fatal case of the disease. The satellite virus, HDV, has a single-stranded RNA genome. It can be found in both positive and negative forms in liver cells. Both the genomic and antigenomic sequences, as the positive and negative forms are called, have regions that fold into an active ribozyme, which can catalyze RNA cleavage and ligation. Unlike plant viroids, HDV also has an open reading frame that encodes a protein called the delta antigen. Delta antigen plus coat proteins from hepatitis B virus are required to package HDV into small spherical particles. These can be spread from cell to cell, and from person to person via bodily fluids such as saliva and semen.

 

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