Transcript Ribozymy - Univerzita Karlova v Praze

Ribozymes
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Ribozyme:

RNA possessing catalytic activity

Increases the rate and specificity
of:

phosphodiester bond
cleavage

peptide bond synthesis

Widespread occurrence in nature
– from viruses to humans

In 1989, Nobel Prize in chemistry has been awarded to Sidney Altman
and Thomas Cech for their discovery that RNA in living cells is not only a
molecule of heredity but also can function as a biocatalyst“
S. Altman
T. Cech
Naturally occurring ribozymes
Ribozyme x protein enzyme

Structural features affect how RNA can function:

RNA contains only 4 unique nucleotide bases compared to 20 AA
found in proteins ( small repertoire of functional groups in RNA)

high density of negative charges

localization of bases in the interior of duplexes ( x amino acid side
chains are directed outward from the polypeptide backbone)

Nevertheless, the mechanisms of catalysis are diverse and exploit:

metal ions

acid-base mechanism, e.g. using nucleobases

small molecule metabolite as a cofactor

substrate (e.g. tRNA) assistance
Usually, ribozyme combines several of these strategies
Ribozyme & protein enzyme

The catalytic strategies appear to be similar: RNA as well as protein
enzymes use acid-base groups and metal ions to activate
nucleophiles and to stabilize developing charge on the leaving group

Ribozyme also requires formation of a specific secondary and tertiary
structure of RNA (by base-pairing of complementary regions); specific
primary structure of certain regions is also necessary

Some ribozymes can speed up the rate of reaction 103-1011 times
(HDV ribozyme cleaves the phosphodiester bond as fast as RNase)
1. Metalloribozymes
a) Ribonuclease P

RNase P catalyzes site-specific hydrolysis of precursor tRNA which is
essential for the formation of mature tRNA

Catalytic activity depends on the presence of divalent cations (Mg2+,
Mn2+)

Large ribozyme, composed of both RNA and protein(s); however, RNA
moiety alone is the catalyst
1. Metalloribozymes
b) Self-splicing introns

Large introns (> 200 nucleotides) that are able to splice-out themselves

In bacteria as well as eukaryotes (e.g. pre-RNA of protozoan
Tetrahymena, primary transcripts of the mitochondrial genes of yeast
and plants…)
Splicing

Introns = segments of noncoding RNA that are interspersed among
the regions of mRNA that code for protein (exons)

Prior to translation, introns must be removed to form a mature mRNA
Genomic
DNA
promotor
region
exon 1 intron 1 exon 2 intron 2 exon 3 intron 3
transcription
Pre-mRNA
1
2
3
splicing
Spliced mRNA
1
2
3
Self-splicing x splicing

Unlike common introns, self-splicing introns can splice themselves out
of pre-mRNA without the need for the spliceosome (complex of RNA
and proteins/enzymes, e.g. helicases)

Although self-splicing introns can remove themselves from RNA in the
absence of any protein in vitro, in many cases in vivo, self-splicing
proceeds in the presence of certain proteins that increase the efficiency
of splicing (e.g. stabilize the correct structure of RNA)

Self-splicing introns mediate only one round of RNA processing (unlike
protein enzymes)
Self-splicing introns:



group I introns: self-splicing is initiated by the nucleophilic attack of
3´-OH of an exogenous guanosine (bound by hydrogen bonds) on
the phosphodiester bond
group II introns: nucleophile attack is realized by 2´-OH of a specific
adenosine within the intron
Metal ions (Mg2+, Mn2+) are proposed to:

promote the formation of the correct active site structure

correctly position the substrate

activate the nucleophile by deprotonating the 2´-OH of guanosine

stabilize the negative charge
Group I introns

3´-OH of an exogenous G attacks the phosphodiester bond at the
5´splice site; this bond is being cleft, G fuses to the 5´end of the intron
…1st transesterification

The freed 3´-end of the exon attacks the bond at the 3´splice site; this
fuses the 2 exons and releases the intron... 2nd transesterification
Group I introns
Group II introns
G nucleotide
binding site
exon 1
G attacks the
phosphodiester bond
at the 5´splice site
exon 2
cleavage between 3‘ end of
exon and 5‘ end of intron
terminal 3‘OH of exon 1
attacks and cleaves the
phosphodiester bond
at the 3‘ splice site
a new bond is formed
between the two exons,
intron is released
p…phosphate
internal
adenosine
internal A attacks the
phosphodiester bond
at the 5´splice site
The importance of being folded:
5´-site of
splicing

site recognized
by guanosine &
site of the first
attack
base-pairing
Specific primary, secondary, and tertiary structure is
necessary for:

recognition of the
guanosine binding site

recognition of the sites
of splicing (attack)
guanosine
binding site
RNA hairpin loop
3´-site of
splicing
RNA Hairpin
backbone
bases in the interior
Group I introns as real enzymes

Self-splicing introns mediate only one round of RNA processing (unlike
protein enzymes)

BUT: once a group I intron has been spliced out, it can act as a real
enzyme: it can repeatedly recognize a complementary sequence of
another RNA molecule (by the internal guide sequence, IGS), attack it
by 3´-OH of the bound G nucleotide, and catalyze its cleavage
RNA substrate
(group 1 intron after
being spliced out)
ribozyme attacking
the RNA substrate
Potential therapeutic use of articifial
group I introns

We can (in vitro) change the IGS, and thus generate tailor-made
ribozymes (ribonucleases) that cleave, i.e. destroy, RNA molecules of
our choice…candidate method for human therapy

Currently: synthetic ribozyme that destroys mRNA encoding the
receptor of Vascular Endothelial Growth Factor (VEGF) is being
readied for clinical trials. VEGF is a major stimulant of angiogenesis,
and blocking its action may help starve cancers of their blood supply.
2. Small ribozymes
of viroids and satellites



Hammerhead
Hairpin
HDV (hepatitis delta virus) ribozyme

Satellites: small RNA viruses or RNA molecules; their multiplication
depends on the mechanisms of a host cell and on the co-infection of a
host cell with a helper virus

Ribozyme is a part of a larger RNA (viroid or satellite) that is being
replicated by host RNA-polymerases

The product of the replication is being self-cleft (by ribozyme activity)
into unit-length RNA molecules
cyclic
phosphate!

Nucleophilic attack of a 2´-OH on the neighbouring 3´-phosphate,
forming 2´-3´ cyclic phosphate

Probably an acid-base mechanism: 2´-OH is activated for a nucleophilic
attack by abstraction of a proton by a basic group (B). Another proton is
donated (by an acid, A) to stabilize the developing negative charge on
the leaving group oxygen (O5´).

In HDV: cytosine (=NH+–) acts as an acid to protonate the leaving group
and a divalent metal ion activates the nucleophile
Hammerhead ribozyme

Hammerhead and hairpin ribozymes can be found in several satellite
RNAs associated with RNA plant viruses (e.g. tobacco ringspot virus)
X

HDV is a human pathogen: co-infection of HDV with HBV is more
severe than infection of HBV alone
3. Riboswitches

Elements of bacterial mRNA that control gene expression via binding
of small molecules (coenzymes, amino acids, nucleobases)

GlmS ribozyme: located in the 5´-untranslated region of mRNA
encoding glucosamine-6-phosphate (GlcN6P) synthetase; in the
presence of GlcN6P(product), it cleaves its own mRNA, which
downregulates the production of the synthetase
riboswitches may have functioned as
metabolite sensors in primitive organisms
Mechanisms of riboswitch-catalyzed reactions

A) „conformational“ – metabolite binding induces a conformational
change in RNA that affects transcription termination/translation initiation

B) „chemical“ – GlmS: GlcN6P amine might serve as an acid to activate
the leaving group cleavage (of the bond in orange):
4. Ribosome is a ribozyme

Peptidyl transferase = ribozyme
translation

Peptidyl transferase activity can be enhanced by protein L27,
however, even in the absence of this protein, reduced activity can still
be observed

Although this protein facilitates peptide bond formation, it is not
essential for peptidyl transferase activity
How does RNA catalyze
peptide bond formation?

Hypotheses:

Base-pairing between the CCA end of tRNAs in the P and A sites
and 23S rRNA help to position the -amino group of aminoacyltRNA to attack the carbonyl group of the growing polypeptide

Proton transfer from the amino group of aminoacyl-tRNA via 2´-OH
of adenosine (from the terminal CCA of tRNA in the P-site) to its O3´
(accompanied by peptidyl (-CO-R) transfer to aminoacyl-tRNA):
O3´
„RNA World“ hypothesis

RNA initially served both as the genetic material and the catalyst; later,
catalytic functions of many RNA molecules were taken over by proteins

Cationic clays such as montmorillonite can promote the polymerization
of RNA-like monomers into „RNA“ chains

RNA is the primary substance of life, DNA and proteins are later
refinements

Cofactors used by ribozymes include e.g.: vit. B12, FMN, glucosamine6-phosphate. Some of them are used by protein enzymes for oxidation,
reduction, C-C bond formation 

Were also RNA molecules capable of something like this?

And have some of them persisted up to now?
Why do we have protein catalysts?

Group I intron active site is mechanistically equivalent to DNA and RNA
polymerases  what selective pressure led to the current protein-based
system for replication and transcription?

The reason might be greater

fidelity

processivity

reaction rates

functional repertoire (provided by 20 AA)