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. Metalloribozymesa) 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. Metalloribozymesb) 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

GenomicDNA

Pre-mRNA

Spliced mRNA

exon 1 exon 3promotor

region intron 1 exon 2 intron 2 intron 3

transcription

1 2 3

splicing

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)

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

Self-splicing introns:

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

G nucleotidebinding site

exon 1 exon 2

Group I introns Group II introns

p…phosphate

cleavage between 3‘ end of exon and 5‘ end of intron

terminal 3‘OH of exon 1 attacks and cleaves the

phosphodiester bondat the 3‘ splice site

a new bond is formedbetween the two exons,intron is released

internaladenosine

G attacks thephosphodiester bond

at the 5´splice site

internal A attacks thephosphodiester bond at the 5´splice site

The importance of being folded:

Specific primary, seconda-ry, and tertiary structure is necessary for:

recognition of the guanosine binding site

recognition of the sites of splicing (attack)

guanosinebinding site

3´-site of splicing

base-pairing

5´-site ofsplicing

site recognizedby guanosine & site of the first attack

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

(group 1 intron after being spliced out)

RNA substrate

ribozyme attackingthe 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

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

cyclicphosphate!

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 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)