Ribozymes

1. Ribozymes mirka.rovenska@lfmotol.cuni.cz

2. 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

3. In 1989, Nobel Prize in chemistry has been awarded to Sidney Altman and Thomas Cechfor 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

4. Naturally occurring ribozymes

5. 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

6. 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)

7. Metalloribozymesa) Ribonuclease P • RNase P catalyzes site-specific hydrolysis of precursor tRNAwhich is essential for the formation of mature tRNA • Catalytic activity depends onthe presence of divalent cations (Mg2+, Mn2+) • Large ribozyme, composed of both RNA and protein(s); however, RNA moiety alone is the catalyst

8. 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…)

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

10. 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 proteinin 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)

11. 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

12. 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... 2ndtransesterification

13. Group I introns Group II introns G nucleotide binding site internal adenosine exon 2 exon 1 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

14. The importance of being folded: site recognized by guanosine & site of the first attack • Specific primary, seconda-ry, and tertiary structure is necessary for: • recognition of the guanosine binding site • recognition of the sites of splicing (attack) 5´-site of splicing base-pairing guanosine binding site 3´-site of splicing RNA hairpin loop

15. RNA Hairpin backbone bases in the interior

16. 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

17. RNA substrate (group 1 intron after being spliced out) ribozyme attacking the RNA substrate

18. 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.

19. 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

20. cyclic phosphate! • Nucleophilic attack of a 2´-OH on the neighbouring 3´-phosphate, forming 2´-3´ cyclic phosphate • Probably anacid-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 theleaving group oxygen (O5´). • In HDV: cytosine (=NH+–) acts as an acid to protonate the leaving group and a divalent metal ion activates the nucleophile

21. Hammerhead ribozyme

22. 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

23. 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

24. 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):

25. 4. Ribosome is a ribozyme • Peptidyl transferase = ribozyme translation

26. 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

27. 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´

28. „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?

29. 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)