MAGNESIUM AND CATALYTIC RNA 239
connected the fl anking exon sequences. In Section 2.3.5 , introns were defi ned
as sections of DNA within a gene that do not encode part of the protein that
the gene produces. Usually, introns are spliced out of the mRNA that is tran-
scribed from the gene before it is translated. Exons were described as regions
of DNA within a gene that are not spliced out from the transcribed RNA and
are retained in the fi nal mRNA molecule. Most, but not all, exon regions are
translated to protein. (See Section 2.3.5.) With experiments carried out in vitro
duplicating the activity seen in vivo inTetrahymena pre - rRNA cells, the ribo-
zyme chemistry taking place was described as follows: (1) Highly purifi ed
pre - rRNA (precursor ribosomal RNA) was mixed with guanosine or guano-
sine triphosphate, GTP, in a solution containing magnesium ions; (2) precise
cutting and joining of phosphodiester bonds took place; and (3) during the
process the added guanosine became covalently linked to the 5 ′ end of the
excised intron.^3 After a year ’ s work attempting to inactivate the splicing
reaction with protein - destroying treatments, the researchers decided to
follow another hypothesis — that the RNA was providing the active site for
its own splicing. This hypothesis was confi rmed by experiments reported in
reference 2.
An entirely new fi eld of catalytic biochemistry would not have developed
if the splicing found forTetrahymena pre - rRNA (classifi ed as a group I intron)
had been the only example of a ribozyme. In 1983 and 1984, however, it was
revealed that RNase P (ribonuclease P) was also a ribozyme. RNase P pro-
cesses the 5 ′ ends of transfer RNA (tRNA) precursors in all organisms. Ribo-
nuclease P (RNase P) is a ubiquitous and essential ribonucleoprotein (RNP),
containing both RNA and protein segments. It has been found that the RNA
subunit by itself can accomplish the required chemistry under conditions of
elevated counterion concentration in vitro.^4 Introns in fungal mitochondrial
mRNA and rRNA (called group I introns) exhibit RNA splicing that is protein -
dependent. The proteins in these systems are called maturases. It was found
thatcis - acting mutations in these introns prevented splicing, indicating that
the RNA structure was important. The reference 4 and 5 publications made a
key connection: The group I introns have secondary structure elements that
are conserved not only with each other but also withTetrahymena pre - rRNA
itself. This led to the hypothesis that the group I introns would be self - splicing
using the same mechanism asTetrahymena pre - rRNA.^5 Work on bacterio-
phage T4 mRNA introns showed that they too were self - splicing tRNAs, con-
tradicting earlier predictions that RNA splicing was confi ned to eukaryotic
species.^6 Group II self - splicing RNAs differ from group I examples in both
RNA structure and splicing mechanism.^7 The hammerhead ribozyme was
discovered as the fi rst small self - cleaving RNA motif.^8 The hammerhead ribo-
zyme catalyzes self - cleavage at a rate some 10^6 - fold greater than that of uncat-
alyzed non - sequence - specifi c RNA self - cleavage. The hammerhead ribozyme
will be discussed in more detail in Section 6.2.4.
Structure – function relationships were established for ribozymes by fi rst
considering how their tertiary structure must be dependent not only on the