EXPERIMENTS:

Thomas cech in 1983 worked to find out the types of RNAs involved in splicing of introns of
tetrahymena. They used standards biochemical fraction methods and found that splicing ability of that
factor by treating with proteases is not finished. It means that no proteins are present in that factor. Then
he took other factors and fractions with phenol in aqueous condition. Phenol is not soluble in water due to
which two layers are formed when the whole mixture was shaken.
RNA preferably soluble in water while protein prefer to soluble in phenol. In this way both
components can be separated. He took aqueous phase which contain pure RNA. He put this in the
medium containing precursors RNA. It was found that splicing took place. By action of proteases heat
treatment by phenolic separation was found that this factor which splice precursor RNA does not require
any protein unit to catalyze splicing. It can individually splice introns. This factor without protein cannot
catalyze splicing in primary that is linear structure but it can do splicing in secondary structure. Further
confirmation was given to this idea by Zang and Cech.
ZANG AND CECH:
In 1986 introducing a particular rDNA in a plasmid by genetic engineering. It was found that
rDNA in plasmid was still transcribing with bacterial RNA polymerase and was still able to splice itself
and splicing reaction was only requiring:
1. Mg
2. ATP
3. Guanosine donar like GMP,GDP,GTP
4. rDNA precursor
5. secondary structure of RNA
This structure was named as ribozymes that is a molecule in which the activation energy for a
chemical reaction is lower by RNA and not by proteins as in enzyme. It was found that ribozyme does not
require ATP to catalyze splicing. It lower the energy of activation itself. Zang and Cech have
demonstrated that excised circular intron has catalytic even after its splicing is completed. So it can act as
an enzyme self splicing introns are also found in mitochondria.
In 1986 it was confirmed that ribozymes are chemically only RNA which has catalytic ability.
This discovery changed the idea that all enzymes are proteins in nature. Now it is said that most enzymes
are proteins in nature but some enzymes are RNA in nature. It also changed the direction of thoughts that
origin of life in universe might not involve d proteins and only RNA had done this job.

RIBOZYME:
A ribozyme is an RNA molecule with a well defined tertiary structure that enable it to
catalyze a chemical reaction. Ribozyme means ribonucleic acid enzyme. It may also be called an RNA
enzyme or catalytic RNA. Many natural ribozyme catalyze either the hydrolysis of one of their own
phosphodiester bonds (self cleaving ribozyme) or the hydrolysis of bonds in other RNAs. Some have
been found to catalyze the aminotransferase activity of the ribosome. Examples of ribozymes include the
hammer head ribozyme the US ribozyme and the hairpin ribozyme. Investigators are studying the origin
of life has produced ribozyme in the laboratory that are capable of catalyzing their own synthesis and are
very specific condition ribozyme. Mutagenesis and selection has been performed resulting in isolation of
improved variants of the round -18 polymerase ribozyme from 2001 is able to add up 10 to 20 nucleotides
to a primer template in 24 hours. Until it decomposes by hydrolysis of its phosphodiester bonds. The
ribozyme can add up to 45 nucleotides with a fidelity of 0.0083 mutation nucleotide.
KNOWN RIBOZYMES:
Naturally occurring ribozyme include:
1. peptidyl transferase 23s rRNA
2. RNAs
3. Group 1 and group 2 introns
4. GTR branching ribozyme
5. Leadzyme although initially evented in vitro natural example have been found
6. Hairpin ribozyme
7. Hammer head ribozyme
8. HDV ribozyme
9. Mammalian CPEB ribozyme
10. VS ribozyme
11. Glms ribozyme
12. CoTc ribozyme
APPLICATIONS:
A type of synthetic ribozyme directed against HIV RNA called gene shears has been developed
and has entered clinical testing for HIV infection.
SPLICING IN PROKARYOTES:
Some sort of splicing also take place in prokaryotes for example in bactariophage T4, these are
present to genes containg one intron one gene is for thymidylate synthase and other for ribonucleotite
reductase. These enzymes are involved in the bio synthesis of deoxyribonucleotides for DNA replication.
When T4 infects host that is E.coli significance of splicing is doubtful but it occur always in eukaryotes
and some time it occur in prokaryotes. So its significance would be interesting but yet not fully
discovered.