Ribozymes mirka.rovenska@lfmotol.cuni.cz

Ribozyme: RNA possessing catalytic activity Increases the rate and specificity of: phosphodiester bond cleavage peptide bond synthesis Widespread occurence in nature – 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 Structural features affect how RNA can function (due to influence on potential contacts and tertiary structure): RNA contains only 4 unique nucleotide bases compared to 20 amino acids 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 of the RNA strand); specific primary structure of particular 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 the protein enzyme RNase)

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(s); however, RNA moiety alone is the catalyst

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 exon 1 exon 3 promotor region intron 1 exon 2 intron 2 intron 3 transcription 1 2 3 splicing Pre-mRNA Spliced mRNA

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 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: attack of an exogenous guanosine 3´-OH of an exogenous guanosine 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 bond at the 3´splice site; this fuses the two exons and releases the intron...2nd transesterification (see 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 5´-site of splicing base-pairing A specific primary, secon-dary, and tertiary structure is necessary for: recognition of the guanosine binding site recognition of the sites of splicing (attack) guanosine binding site 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

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 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

Similar to reactions catalyzed by protein RNases cyclic phosphate! Nucleophilic attack of a 2´-OH on the neighbouring 3´-phosphate, forming 2´-3´ cyclic phosphate Probably: a general acid-base mechanism: 2´-OH is activated for the 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´). Similar to reactions catalyzed by protein RNases 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 or translation initiation B) „chemical“ – in the case of 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 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 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): According to this model, tRNA is essential for catalysis!!! O3´

„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 with distinct sequence selectivity  RNA is the primary substance of life while DNA and proteins are later refinements Cofactors used by ribozymes include e.g.: vit. B12, FMN, glucosamine-6-phosphate, S-adenosyl methionine. Some of these cofactors 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 (protein enzymes) DNA and RNA polymerases (organization of metals is identical)  what selective pressure led to the current protein-based system for replication and transcription? The reason is probably greater fidelity, processivity, and reaction rates in protein-based systems