Ribozymes

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 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 (Mg 2+, Mn 2+ ) 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 DNA Pre-mRNA Spliced mRNA exon 1 exon 3 promotor region intron 1exon 2intron 2intron 3 transcription 123 splicing 123

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 (Mg 2+, Mn 2+ ) 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 …1 st 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... 2 nd transesterification

G nucleotide binding site exon 1 exon 2 Group I intronsGroup 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 bond at the 3‘ splice site a new bond is formed between the two exons, intron is released internal adenosine G attacks the phosphodiester bond at the 5´splice site internal A attacks the phosphodiester 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) guanosine binding site 3´-site of splicing base-pairing 5´-site of splicing site recognized by 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 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

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 cyclic phosphate!

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. B 12, 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)