Page 82 - Academic Press Encyclopedia of Physical Science and Technology 3rd Molecular Biology

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254 Ribozymes

but also a phenotype. The RNA catalyzed reactions in- Despite all the evidence for self-splicing in vitro,itis
clude self-cleavage, or trans-cleavage reactions, ligation, clear that splicing in vivo requires protein factors. Even the
and trans-splicing. These observations have led to specu- Tetrahymena IVS, which at low levels of Mg 2+ splices
lation that RNA might have been an early self-replicating efficiently in vitro, is splicing at a rate of about 50-fold
molecule in the prebiotic world. Evidence supporting this less than the level estimated for splicing in vivo. Proteins
notion comes from the fact that group I introns can exhibit therefore aid in the folding of these complex RNAs to
RNA polymerase-like activities under certain conditions. allow the self-splicing reaction to occur.
In addition, the catalytic core of group I introns shares ho- Self-splicing is, by definition, an intramolecular event,
mology with several small satellite RNAs associated with and the intron is therefore not acting as a true enzyme.
plant viruses, which are also homologous to the human However, the catalytic activity found within the conserved
hepatitis delta virus (HDV), suggesting a common and core, with a small deletion, can be dissociated into distinct
ancient origin. active enzyme and substrate molecules. Cleavage at the
5 - and 3 -splice sites of group I introns can also occur

slowly in the absence of a guanosine cofactor, due to the
II. GROUP I AND GROUP II INTRONS sensitivity of these sites to base hydrolysis which gener-
ates cleaved products consistent with the splicing reaction
Introns are noncoding sequences that interrupt parts of (3 -OH and 5 -P) but unusual for the hydrolysis reaction.

genes. When a pre-mRNA is transcribed from the gene, The rate of this type of hydrolysis at the splice sites is much
introns need to be removed to give rise to mature mes- greater than expected (10-fold higher), implying that the
sengers that will become a template for protein synthe- folded RNA structure influences the susceptibility of cer-
sis. Introns are removed by a process of cleavage-ligation tain phosphodiester bonds to alkaline hydrolysis.
called splicing. Generally, splicing requires a multicom- Shortened versions of the Tetrahymena IVS (L-19 IVS
plex of proteins and RNA. When introns were first discov- and L-21 Scal IVS) have been shown to be true enzymes
ered in nuclear genes, it was noted that all of their DNA in vitro, for example, as a restriction endoribonuclease and
sequences began with a GT and ended with an AG dinu- as a template-dependent polymerase.
cleotide (Chambon’s rule). Certain introns isolated from
ribosomal or organellar genes, however, did not follow
B. Group II Introns
this simple rule, and DNA sequence comparisons led to
the classification of these introns as either group I or group Group II introns present a relatively restricted distribu-
II on the basis of phylogenetically conserved sequence tion; they have been found in plant and fungal mtDNAs
homologies and secondary structures. Some examples of and comprise the majority of the introns in chloroplasts.
group I and II introns are capable of self-splicing in vitro It has also been demonstrated that some members of the
in the absence of protein. Those catalytic RNAs produce group II introns can self-splice in vitro. The unimolecu-
5 -phosphate and 3 -OH termini on the reaction products. lar reaction was shown to be Mg 2+ dependent, requiring

spermidine, having a temperature optimum of 45 C, and
◦
having a pH optimum of between 6.5 and 8.5. They dif-
A. Group I Introns
fer from group I introns by the structure of their catalytic
Group I introns are widely distributed in fungal mitochon- core and the intermediate and end products of splicing
dria, chloroplasts, rRNA genes of protists, T-even phages, which involve a lariat structure. Again, the reaction con-
andthegenomesofeubacteria.GroupIintronself-splicing sists of two transesterifications. Group II introns splice by
(in vitro) in the absence of proteins was first observed for way of two successive phosphate transfer reactions. In the
the intervening sequence (IVS, intron) of the nuclear 26S first step, the 2 -OH group of an intramolecular branch

rRNA gene in Tetrahymena thermophila. point adenosine attacks the phosphodiester bond at the
Group I splicing proceeds by two consecutive trans- 5 -splice site (creating a 2 ,5 -bond), producing the free

esterification reactions. These reactions are initiated by a 5 -exon and a splicing intermediate, the intron-3 -exon.

nucleophilic attack by the 3 -hydroxyl of a guanosine (or The second step involves cleavage at the 3 -splice site by

a phosphorylated derivative: GMP, GDP, or GTP) at the the 3 -OH of the 5 -exon. Simultaneously, the exons are

phosphodiester bond between the 5 -exon and the intron ligated and the intron lariat, with a 2 ,5 -phosphodiester

(5 -splice site). The new 3 -hydroxyl group of the 5 -exon bond, is released. Base-pairing interactions between se-

then initiates a second nucleophilic attack, this time on the quences known as the exon binding site (EBS) and the

phosphodiester bond between the 3 -exon and the intron intron binding site (IBS) hold the splice sites in close prox-

(the 3 -splice site). This results in ligation of the exons and imity. This ability of group II introns to specifically bind
excision of the intron. the 5 -exon has been exploited to encourage the intron to