Ribozymes mirka.rovenska@lfmotol.cuni.cz

Ribozyme: Catalytic RNA molecule Enhances the speed and specificity of: phosphodiester bond cleavage peptide bond synthesis Naturally occurring – from viruses to humans

In 1989, Nobel Prize in chemistry „has been awarded to Sidney Altman, USA and Thomas Cech, USA for their discovery that RNA (ribonucleic acid) 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 RNA contains only four unique nucleotide bases (compared to 20 different amino acids found in proteins)  limited range of catalysis mechanisms (compared to proteins) High density of negative charges and localization of bases in the interior of base-paired duplexes also affect how RNA can function (due to influence on potential contacts and tertiary structure)

Ribozyme & protein enzyme Nevertheless, the catalytic strategies appear to be similar: RNA and protein enzymes both 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 of the RNA strand); certain primary structure of particular regions is also necessary Many ribozymes can speed up the rate of reaction 103-1011 times (HDV ribozyme cleaves the phosphodiester bond as fast as the protein enzyme RNase

1. Metalloribozymes a) Ribonuclease P RNase P catalyzes site-specific hydrolysis of precursor tRNA …essential for the formation of mature tRNA In bacteria as well as eukaryotes (including humans) Catalytic activity depends on the presence of divalent cations (Mg2+, Mn2+) Large ribozyme, composed of both RNA and protein, however, RNA moiety alone is the catalyst

Hydroxide ion or metal-coordinated hydroxide attacks the phosphate atom at the same time as the phosphate-oxygen bond begins to break Metal ion(s) have been proposed to play multiple roles, e.g. activation of the water nucleophile, stabilization of the developing negative charge

1. Metalloribozymes b) Self-splicing introns Large introns (> 200 nucleotides) that are able to splice-out themselves In bacteria as well as eucaryotes (e.g. pre-RNA of protozoan Tetrahymena, primary transcripts of the mitochondrial genes of yeast, …)

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 promoter 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) However, although self-splicing introns can remove themselves from RNA in the absence of any protein, in many cases in vivo, self-splicing proceeds in the presence of certain proteins that increase the efficiency of splicing (stabilize the correct structure) Self-splicing introns mediate only one round of RNA processing – despite protein enzymes

Self-splicing introns: group I introns: self-splicing is initiated by nucleophilic attack of an exogenous guanosine (bound by hydrogen bonds) on the phosphodiester bond group II introns: nucleophile attack realized by 2´-hydroxyl group 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, and stabilize the negative charge

Group I introns: attack of an exogenous G 3´-OH of an exogenous G attacks the phosphodiester bond at the 5´splice site; this bond is being cleft and G fuses to the 5´end of the intron…first transesterification Then, the freed 3´-end of the exon attacks the ester bond at the 3´splice site; this fuses the two exons and releases the intron...second transesterification (next picture)

Group I introns Group II introns internal A attacks the G nucleotide binding site internal adenosine exon 1 exon 2 internal A attacks the phosphodiester bond at the 5´splice site G attacks the phosphodiester bond at the 5´splice site 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

The importance of being folded: site recognized by guanosine & site of the first attack Certain primary, secondary and tertiary structure are necessary for: recognizing of the guanosine binding site recognizing of the sites of splicing (attack) 5´-site of splicing base-pairing guanosine binding site 3´-site of splicing RNA hairpin loop

RNA Hairpin – 3D backbone bases in the interior

Self-splicing introns mediate only one round of RNA processing – despite protein enzymes BUT: once a group I self-splicing intron has been spliced out, it can act as a real enzyme (ribozyme): 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

ribozyme attacking the RNA substrate 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 the mRNA encoding a 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) Satellite: a small subviral agent lacking genes encoding for the enzymes catalyzing its replication; multiplication of a satellite depends on the mechanisms of a host cell as well as 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 (ribozyme activity) into unit-length RNA molecules These can be then ligated into circles (again by ribozyme activity)

Similar to reactions catalyzed by protein RNases cyclic phosphate! Catalyze the nucleophilic attack of a 2´-hydroxyl on the neighbouring 3´-phosphate, forming 2´-3´ cyclic phosphate Reaction is proposed to be catalyzed by a general acid-base mechanism: 2´-hydroxyl adjacent to the scissile phosphate is activated for the nucleophilic attack by abstraction of a proton by a basic group. Concurrently, a proton is donated to stabilize the developing negative charge on the leaving group oxygen Similar to reactions catalyzed by protein RNases

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. Ribosome is a ribozyme Peptide bond formation is catalyzed by the 23S rRNA component of the large subunit of a ribosome: translation

Peptidyl transferase activity can be enhanced by a certain 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 peptide transferase activity

How does RNA catalyze peptide bond formation? 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 Yet another catalytic contribution of RNA is likely: it has been proposed (not confirmed) that 3´oxygen of the terminal adenosine in the CCA terminal triplet of peptidyl-tRNA extracts a hydrogen from 2´-OH and 2´oxygen, in turns, extracts a hydrogen from the amino group attacking the carbonyl

„RNA World“ hypothesis RNA initially served both as the genetic material and the cellular catalyst; during evolution, the 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 Even in the absence of enzymatic catalysts, single-stranded RNAs may have been able to copy strands of RNA through template-directed polymerization using activated nucleotides In vitro, novel (unnatural) catalytic RNAs have been discovered exhibiting a wide repertoir of catalytic activities and capable also of using only 3 nucleotides (A, G, U)