1. RIBOZYMES
NUCLEIC ACIDS THAT ENDED THE LONG BELIEVED CATALYTIC MONOPOLY OF PROTEINS.

2. A RIBOZYME IN VIEW

3. RIBOZYMES ARE CATALYTIC RNA
The term ‘ribozyme’ can be broken down to the two components, ribo and enzyme, both of which are very well known to us.
‘Ribo’ ,as we understand, denotes Ribonucleic Acid or RNA. Enzymes, as we define them, are biocatalysts. They are macromolecules that carry out catalysis.
Therefore, catalytic RNAs are called ribonucleic acid enzymes or ribozymes.
Catalytic RNA is a rather recent discovery and one that shook the biological world, which till then had believed that ‘all enzymes are proteins’.

4. HOW DOES RNA CARRY OUT CATALYSIS?
The most necessary requirement of an enzyme or any other catalyst is a surface on which the reaction can be catalysed and carried out.
The surface must possess unique contours and chemical properties so that the substrates can bind and react.
Substrate binding is important as it is the binding energy of the substrates that lowers the activation energy of the enzyme catalysed reaction as compared to the non-catalysed reaction.
Lower activation energy corresponds to a faster reaction.
RNA molecules can carry out catalysis because they can provide a variety of such surfaces.

5. RNA CAN FORM COMPLEX SECONDARY AND TERTIARY STRUCTURES
The ability of forming extensive secondary and complex
tertiary structures is what provides RNA with it’s ability
to form a vast array of different catalytic surfaces.

6. RNA SECONDARY STRUCTURES
The red lines indicate Watson-Crick base pairing.

7. COMPLEX RNA STRUCTURES OCCUR DUE TO...
The fact that RNA is almost always single stranded, because save in the few cases of viruses, RNA is not the genetic material but a product of transcription. Single stranded RNA can fold, loop in and out, form hairpin, cruciform and other secondary/tertiary structures due to paining between the exposed bases.
RNA also exhibits a special kind of base pairing, a G=U pair, an example of non Watson-Crick base pairing, which plays a significant role in the formation of complex structures.

8. G.U BASE PAIRING
G.U base pairing in tRNA

9. RNA TERTIARY STRUCTURES
These structures bring together distant parts of the same
RNA molecule or two different RNA molecules much in the
same way tertiary structures in proteins do.

10. RNA AS ENZYMES
We have already explored the fact that RNA molecules can function as enzymes because the higher ordered structures they can adopt help to provide a wide variety of surfaces for catalysis of reactions.
Therefore, an RNA molecule with appropriately folded conformation can act as enzymes.
Like many enzymes, most of these ribozymes can function by positioning a metal at the active site. This greatly enhances the range of reactions catalysed and hence ribozymes carry out more diverse reactions than can be solely accounted for by the limited chemical activity of the polynucleotide chain.

11. RNA AS ENZYMES
Ribozymes vary greatly in size. A self splicing group I intron may have 400 nucleotides, whereas the hammerhead ribozyme , found in virusoids, consists of two RNA strands with 41 nucleotides in all.
The three-dimensional structure of the ribozyme is very important as in protein enzymes . The enzymatic activity is lost upon heating, adding denaturing agents or complementary oligonucleotides as all of these treatments disrupt the normal base pairing pattern and destroy the structure.
Ribozymes can also be inactivated by mutations in the normal sequence of the bases in the polynucleotide, as it changes the structure.

12. MOST COMMON RIBOZYMES ARE ENZYMES CONCERNED WITH RNA METABOLISM
These RNA molecule catalyze the cleavage of
a second RNA at a specific site. This is a
common phenomenon in RNA processing.
Ribozymes are involved in mRNA
splicing of Group I and Group II introns,
a process that does not require ATP, in the
form of snRNPs ( small nuclear RiboNucleo-
protein Particles). The RNA component of the
nucleoprotein complex is snRNA. Splicing
reactions require a properly positioned Mg
2+ ion at the active site. Some ribozymes are
self-cleaving and cleave their own
phosphodiester bonds. They are consumed
by their own reactions.

13. VERSATILITY OF RIBOZYMES
Ribozymes can carry out a host of other reactions like aminotransferase reactions, RNA ligation reactions, transesterification and phosphorylation reactions etc.
Ribozymes have been produced in the laboratory that catalyze their own synthesis, under specific conditions and the presence of RNA Polymerase ribozyme.
Like proteins, RNA can undergo allosteric conformational changes, either in response to small molecules to other RNAs.
There is the instance of an artificially synthesized ribozyme that can exist in two different conformational states and catalyzes different reactions in these two states.
RNA are extremely versatile molecules that possess considerable biochemical sophistication and the prime testimony to this fact is the rRNA present in the ribosome.
RNA has been demonstrated to catalyze protein conformation changes in prions, much like chaperones.

14. NATURAL AND ARTIFICIAL RIBOZYMES
The most common ribozymes occurring naturally are the ribozymes in the rRNA of ribosomes and in the RNase P complex.
Other widely studied ribozymes include the hairpin ribozyme and the hammerhead ribozymes.
New synthetic ribozymes have been developed in the laboratory. These include RNA molecules capable of self-cleavage.
The techniques used to create artificial ribozymes utilise the concept of Darwinian Evolution. Since RNA molecules are both informational and self replicating, they increase rapidly in number in a pool of macro molecules.
Then, a single type of ribozyme can be isolated and studied by sorting it out through the reaction it catalyzes.
Most of our knowledge about ribozymes comes from such synthesis.

15. THE RNase P COMPLEX CONTAINS A RIBOZYME
The RNase P complex, another
ribonucleoprotein, was what
first led to the discovery of the
ribozyme. It was then being
studied by Sidney Altman. He,
along with Thomas. R. Cech who
was working on self splicing of
mRNA in Tetrahymena
thermophilis, were the first to
report catalytic activity of RNA in
the 1980s. The RNA component of
E.coli RNase P has a secondary
structure as elucidated on the
left.

16. THE STUDY OF RNase P BROUGHT STARTLING REVEALATIONS
Ribonuclease P, a tRNA processing
endonuclease in E.coli can be
dissociated into its two components
1. A 375 base RNA 2. A 20 kD
polypeptide. Initially, it was noticed
that in vitro both components were
necessary to cleave the tRNA
substrate. However, it was
discovered later that a change in
ionic conditions, caused by an
increase in Mg 2+ concentration,
renders the protein component
superfluous. Given alongside are
two representations of Rnase P.

17. HOW IS CATALYSIS POSSIBLE WITHOUT A PROTEIN??
It was then discovered,amidst much awe and wonder that
the RNA alone can catalyze the reaction. The protein
component, though, plays an important role in substrate
binding and increases the velocity of the reaction. Thus,
very curiously, the roles of RNA and protein had reversed
from what had been
speculated initially.
Mutations in either the
RNA or Protein component
renders the complex
catalytically inactive. This
work earned Cech and
Altman the Nobel Prize for
Chemistry in 1990.

18. HAMMERHEAD RIBOZYME
This kind of ribozyme has been studied in virusoids.
Virusoids are certain virus-like elements which have small RNA genomes and are required to be assisted by other viruses in replication and/or packaging.
Some virusoid RNAs include small segments that promote site-specific RNA cleavage reactions associated with replication. These small RNA segments act as ribozymes.
They get their name from their resemblance in shape to the head of a hammer.
Hammerhead ribozymes have been defined and studied separately from the much larger viral genome.

19. HAMMERHEAD RIBOZYME Base pairing between the
Ribozyme and the substrate
with the enzyme shown in
pink and the substrate in
green.
The hammerhead ribozyme catalyzes
RNA cleavage at specific sites. These
cleavage reactions are very vital steps in the viral life cycle. The enzyme
requires Mg 2+ as a cofactor, bound to which it can adopt a reactive
conformation.

20. THE RIBOSOME IS A RIBONUCLEOPROTEIN
The ribosome is the protein synthesizing machinery of the cell.
The ribosome is a ribonucleoprotein particle, i.e., it is composed of ribosomal RNA or rRNA and proteins.
Ribosomes constitute about 25% of the dry weight of a typical prokaryotic cell and are composed of 65% rRNA and 35% proteins. They are 18 nm in diameter.
The prokaryotic ribosome is 70S and is composed of a 50S and a 30S subunit. The eukaryotic cytoplasmic ribosome is 80S and composed of subunits 60S and 40S. S stands for Svedberg or Sedimentation coefficient.

21. THE RIBOSOME IS A COMPLEX ASSEMBLY
In the late 1960s, Nomura et al demonstrated that the
ribosome could be dissociated into its protein and rRNA
components and then reconstituted in vitro to obtain the
intact native conformation with its activity completely
restored. This breakthrough drew the attention of the
scientists towards the components of the ribosome.

22. 50S ribosomal subunit structure of Haloarcula marismortui by Thomas Steitz, Peter Moore, et.al.
• Proteins
• rRNA (bases
white and
backbone
white)
• mRNA
• Protein
• rRNA

23. A Model of a Complete Active Bacterial Ribosome
A – aminoacyl site
P – peptidyl site
E – exit site
A view down into the groove separating the subunits.

24. THE RIBOSOME IS IN EFFECT IS A RIBOZYME
The dawn of the new millennium brought with it the elucidation of the first high resolution image of the ribosome.
Among all the breathtaking observations the standout one was the fact that no protein was found within 1.8 nm of the active site for peptide bond formation.
This caused a shift from the traditional focus on proteins to a new focus on rRNA as the catalytic molecule responsible for peptide bond formation.
Investigators have isolated artificial ribozymes that promote peptide synthesis. Interestingly, all of these contain the conserved sequence 5’-AUAACAGG-3’.

25. STRUCTURE FUNCTION RELATIONSHIP IN RIBOSOMES
Structure of the 50S bacterial
ribosome subunit
The active site for peptide bond
formation, deep within a surface groove
and far away from any protein, is
marked by a bound inhibitor, puromycin
(red).

26. THE RIBOSOME IN EFFECT IS A RIBOZYME
The sequence is a highly conserved sequence found at the
peptidyl transferase active site in the ribosome of all cells.
The high resolution structure thus proved that the
ribosome is not merely a passive platform for translation
but is actually a ribozyme. The role of the proteins in the
is yet undetermined but it is strongly suspected that they
form supportive structures.

27. THE RNA WORLD THEORY The RNA World hypothesis suggests
A solution to the problem of the origin
of a self-replicating molecule and
puts forward the theory that the first
Self-replicating molecules were RNA.
The basis for this theory lies at the
very heart of our discussion –
RIBOZYMES.
The ocean waters or primordial
Soup of primitive Earth gave rise to a
number of complex organic
molecules from simple inorganic
Through simple organic molecules.
One of these happened to be RNA.

28. DUAL ACYTIVITY OF RNA MOLECULES...
The main reason behind
suggesting that RNA might have
been the first self-replicating
molecule is that it has a dual
activity. It is an informational
macromolecule, as seen in some
viruses today where they
constitute the genetic material.
It is also an enzyme, as seen in
ribozymes. The fact that RNA
molecules can direct and catalyze
their own replication would have
been very useful on a primitive
Earth without proteins.

29. ...AND THEIR GRADUAL SPECIALISATON
Replication would not have been
perfect as RNA Polymerases have low
fidelity. Thus, variants will have been
produced. The better variants would
have been more successful in
surviving. DNA could have been one
such variant which would have been
found to be a lot more stable. Thus,
DNA would have stayed on and
replicated. RNA would have also
begun coding for and synthesizing
proteins to increase the efficacy and
speed of replication. The first cell
would thus be formed by enclosing these
contents within a double layer of lipids.

30. EVOLUTION OF DNA GENOME FRON RNA GENOME
Gradually, proteins took over the role of
carrying out cellular functions while the
coding functions were taken by DNA which
came to be the genetic material or the main
information macromolecule. The RNAs were
from then on to continue in their specialised
roles.
The roles played by RNA at present are:
mRNA transcripts link genes and proteins;
rRNA act as ribozymes in protein synthesis;
tRNA acts as the adapter molecule that
forms the polypeptide chain.

31. RNA SORTS THE PARADOX
RNA seems to be the perfect answer to the chicken-egg paradox regarding the origin of self-replicating molecules.
The question as to which among DNA and Protein arose first in evolution is paradoxical because proteins are formed when DNA codes for it. However, for DNA to propagate via replication and maintain itself, proteins are required as they carry out all the cellular processes.
Here, RNA circumvents the problem as it can act both as the chicken and as the egg.
This is possible because RNA is a ribozyme, an information molecule that can catalyze its own replication.
Therefore, it will not be wrong to call RNA a living fossil; the ancestor of all life from a long time back – from the RNA World.

32. THAT’S ALL FOLKS..