Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 290

A ribozyme is an enzyme in which RNA, not protein, carries out catalysis. The first ribozyme discovered was the group I intron of the ciliate Tetrahymena thermophila. Other ribozymes have since been discovered, including RNase P of bacteria, group II self-splicing introns, hammerhead RNAs of viroids and satellite RNAs, and the ribozyme of hepatitis delta virus. These catalytic RNAs are very diverse in size, sequence, and the mechanism of catalysis. For example, the hepatitis delta virus ribozyme (see the figure) catalyzes a transesterification reaction that yields products with 2′,3′-cyclic phosphate and 5′-OH termini. Only an 85-nucleotide sequence is required for activity of this ribozyme, and can cleave optimally with as little as a single nucleotide 5′ to the site of cleavage.

Ribozymes have been essential for producing infectious RNAs from cloned DNA copies of the genomes of (−) strand RNA viruses. Such transcripts often have extra sequences at the 3′ end. By joining the 85-nucleotide ribozyme fragment to upstream sequences, accurate 3′ ends of heterologous RNA transcripts synthesized in vitro can be obtained.

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Crystal structure of the hepatitis delta virus ribozyme. The RNA backbone is shown as a ribbon. The two helical stacks are shown in red and blue, and unpaired nucleotides are gray. The 5′ nucleotide, which marks the active site, is green (PDB file 1cx0).

All hepatitis delta virus RNAs are synthesized in the nucleus by DNA-dependent RNA polymerase II. The ability of this cellular enzyme to copy an RNA template provides a missing link in molecular evolution. This activity supports the hypothesis that an ancestor of RNA polymerase II copied RNA genomes that are thought to have existed during the ancient RNA world. During the course of evolution, enzymes with this property acquired the ability to copy DNA templates, facilitating the transition from RNA to DNA genomes. Today RNA polymerase II can still copy small RNAs such as the genome of hepatitis delta virus.

The switch from mRNA synthesis to the production of full-length (+) strand RNA of hepatitis delta virus is controlled by suppression of a poly(A) signal. Full-length (−) and (+) strand RNAs are copied by a rolling-circle mechanism, and ribozyme self-cleavage releases linear monomers. Subsequent ligation of the two termini by the same ribozyme produces a monomeric circular RNA. The hepatitis delta virus ribozymes are therefore needed to process the intermediates of rolling-circle RNA replication. RNA polymerase II initiates viral mRNA synthesis at a position on the genome near the beginning of the delta antigen-coding region. Once the polymerase has moved past a polyadenylation signal and the self-cleavage domain (Fig. 6.25), the 3′ poly(A) of the mRNA is made by host cell enzymes. The RNA downstream of the poly(A) site is not degraded, in contrast to that of other mRNA precursors made by RNA polymerase II, but is elongated until a complete full-length (+) strand is made. The poly(A) addition site in this full-length (+) strand RNA is not used, perhaps because the delta antigen bound to the rod-like RNA blocks access of cellular enzymes to the poly(A) signal.