Ribozymes

mirka.rovenska@lfmotol.cuni.cz
 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 promotor
region exon 1 intron 1 exon 2 intron 2 exon 3 intron 3
DNA
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

internal
exon 1 exon 2 adenosine

internal A attacks the

G attacks the cleavage between 3‘ end of phosphodiester bond
phosphodiester bond exon and 5‘ end of intron
at the 5´splice site
at the 5´splice site

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
 The importance of being folded:

site recognized
5´-site of by guanosine &
splicing site of the first
attack base-pairing

 Specific primary, seconda-

ry, and tertiary structure is
necessary for:

 recognition of the
guanosine binding site
guanosine
 recognition of the sites
binding site
of splicing (attack)
3´-site of
splicing
RNA hairpin loop
 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)

 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 aminoacyl-
tRNA 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, glucosamine-

6-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)