Transcript RIBOZYMES

ASHA S V
BCH-10-05-05
 Ribozyme, or RNA enzyme, is a RNA molecule that
act as enzymes, often found to catalyze cleavage of
either its own or other RNAs.
 Due to their complex secondary structures and
hairpin/hammer head active centres, RNAs could act
as a catalyst and this idea was proposed by Carl Woese
et al, Thomas R Cech and Sidney Altman were the first
to discover ribozyme.
 It has also been found to catalyze the
aminotransferase activity of the ribosome.
 The RNA catalysts called ribozymes are found in the
nucleus, mitochondria, and chloroplasts of eukaryotic
organisms. Some viruses, including several bacterial
viruses, also have ribozymes.
 Almost all ribozymes are involved in processing RNA.
 They act either as molecular scissors to cleave precursor
RNA chains (the chains that form the basis of a new RNA
chain) or as "molecular staplers" that ligate two RNA
molecules together.
 Although most ribozyme targets are RNA, there is now very
strong evidence that the linkage of amino acids into
proteins, which occurs at the ribosome during translation,
is also catalyzed by RNA. Thus, the ribosomal RNA is itself
also a ribozyme.
Types of ribozymes
 Ribozymes may be classified into natural ribozymes
and artificial ribozymes
 Natural ribozymes include,
 Peptidyl transferase 23S rRNA,RNase P, Group I and
Group II introns, G1R1 branching ribozyme,Leadzyme,
Hairpin ribozyme, Hammerhead ribozyme, HDV
ribozyme, Mammalian CPEB3 ribozyme, VS ribozyme,
glmS ribozyme, CoTC ribozyme
 Artificial ribozymes are synthesised in the laboratory
based on the dual nature of RNAs as a catalyst and an
informational polymer
.
 Synthesis of artificial ribozymes involves the mutation
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of natural ribozymes.
This process is initiated by reverse transcription using
reverse transcriptase. this results in the generation of
various cDNA.
Relatively short RNA molecules that can duplicates
others have been artificially produced in the lab.
This includes a 165-base long RNA and a 189-base long
RNA. these ribozymes could polymerize RNA primers.
A short RNA molecule which mimics one of the two
rRNAs of ribozymes has been synthesised invitro. this
short strand of rRNA catalyzes the synthesis of peptide
bond.
 Hammerhead ribozyme
 Hammerhead RNAs are RNAs that self-cleave via a
small conserved secondary structural motif termed a
hammerhead because of its shape.
 Most hammerhead RNAs are subsets of two classes of
plant pathogenic RNAs: the satellite RNAs of RNA
viruses and the viroids.
 The minimal hammerhead ribozymes
 The minimal hammerhead sequence that is required for
the self-cleavage reaction includes approximately 13
conserved or invariant "core" nucleotides, most of which
are not involved in forming canonical Watson-Crick basepairs.
 The core region is flanked by Stems I, II and III, which are
in general made of canonical Watson-Crick base-pairs but
are otherwise not constrained with respect to sequence.
 The catalytic turnover rate of minimal hammerhead
ribozymes is ~ 1/min . Much of the experimental work
carried out on hammerhead ribozymes has used a minimal
construct.
The crystal structure of a minimal hammerhead
ribozyme
 Structurally the hammerhead ribozyme is composed
of three base paired helices, separated by short linkers
of conserved sequences. These helices are called I, II
and III.
 Hammerhead ribozymes can be classified into three
types based on which helix the 5' and 3' ends are found
in.
 If the 5' and 3' ends of the sequence contribute to stem
I then it is a type I hammerhead ribozyme, and if the
and 3' ends of the sequence contribute to stem III then
it is a type III hammerhead ribozyme.
 Of the three possible topological types both type I and
type III are common. It is not known if examples of the
type II topology are found in nature.
 Hammerhead ribozyme (type I)
 Hammerhead ribozyme (type III)
 The full-length hammerhead ribozyme
 The full-length hammerhead ribozyme consists of
additional sequence elements in stems I and II that
permit additional tertiary contacts to form.
 The tertiary interactions stabilize the active
conformation of the ribozyme, resulting in cleavage
rates up to 1000-fold greater than those for
corresponding minimal hammerhead sequences.
Three-dimensional structure of the full-length
hammerhead ribozyme
 Hairpin ribozyme
 The hairpin ribozyme is a small section of RNA that
can act as an enzyme known as a ribozyme.
 Like the hammerhead ribozyme it is found in RNA
satellites of plant viruses.
 It was first identified in the minus strand of the
tobacco ring spot virus (TRSV) satellite RNA where it
catalyzes self-cleavage and joining (ligation) reactions
to process the products of rolling circle virus
replication into linear and circular satellite RNA
molecules.
 The hairpin ribozyme is similar to the hammerhead
ribozyme in that it does not require a metal ion for the
reaction
 Leadzyme
 Leadzyme is a small ribozyme that was artificially
made using in vitro selection techniques.
 Leadzyme is able to cleave RNA in the presence of
lead
 The structure of leadzyme has been determined by Xray crystallography
 It has been proposed that a naturally occurring
leadzyme occurs in the 5S rRNA and further that this
may be an important mechanism in lead toxicity.
 Hepatitis delta virus ribozyme
 HDV ribozyme is composed of five helical segments
connected by a double pseudoknot.
 Pseudoknot is a kind of teritiary interaction in RNA
which can join distant parts of the same RNA strand.
 It is involved in a self cleavage process needed for
processing the RNA transcripts and it is needeD for
viral replication.
 HDV ribozyme is the fastest known natural self
cleaving RNA.
 The Varkud satellite (VS) ribozyme
 The Varkud satellite (VS) ribozyme is an RNA
enzyme that carries out the cleavage of a
phosphodiester bond.
 The VS ribozyme is composed of 5 helices that form an
H shape (helices II to VI). The first helix (I) contains
the substrate cleavage site in the stem-loop
 Reactions catalyzed by ribozymes
 Ribozymes can catalyze a whole range of reactions
 This includes hydrolysis of their own RNA component
by cleaving the phosphodiester bond[these ribozymes
are called RNA polymerizing ribozymes] as well as
hydrolysis of other RNA strands and aminotransferase
activity of the ribozyme.
 A list of rections catalysed by ribozymes are;
 Hydrolysis of other RNA strands
 Catalysis of self synthesis by templated polymerization
 Self splicing
 DNA and RNA phosphorylation by synthetic ribozyme
 Catalysis of peptidyl transferase reaction during
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translation
Aminotransferase reaction of the ribosome
Catalysis of tRNA maturation
Catalysis of self ligation and self cleavage
Chaperon reactions
 Ribozymes as a cofactors
 RNA can act as a cofactor for amino acid residues.
 Amino acid complexes with RNA molecules during
which the later functions as a cofactor, enhancing or
diversifying the enzymatic capabilities of proteins.
 mRNAs have evolved from RNA molecules which
catalyzes amino acid transfer to them
 Chaperon like ribozymes
 Like chaperon, RNA catalyzes protein folding. Such
RNAs are called chaperon like ribozymes
 Applications
 A type of synthetic ribozyme directed against HIV
RNA called gene shears has been developed and has
entered clinical testing for HIV infection
reference
 Enzymology ;T Devasena
 Functional metabolism regulation and
adaptation;Kenneth B Storey
 Text book of biochemistry ;Thomas M Devlin