Ribozymes.ppt
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Ribozymes
[email protected]
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. 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 (Mg2+, Mn2+)
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
promotor 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)
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
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
Group I introns
Group II introns
G nucleotide binding site
exon 1
G attacks the phosphodiester bond at the 5´splice site
exon 2
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
internal adenosine
internal A attacks the phosphodiester bond at the 5´splice site
The importance of being folded:
5´-site of splicing
site recognized by guanosine & site of the first attack
base-pairing
Specific primary, secondary, and tertiary structure is necessary for:
recognition of the guanosine binding site
recognition of the sites of splicing (attack)
guanosine binding site
RNA hairpin loop
3´-site of splicing
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
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
cyclic phosphate!
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
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 aminoacyltRNA 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, glucosamine6-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)
[email protected]
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. 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 (Mg2+, Mn2+)
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
promotor 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)
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
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
Group I introns
Group II introns
G nucleotide binding site
exon 1
G attacks the phosphodiester bond at the 5´splice site
exon 2
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
internal adenosine
internal A attacks the phosphodiester bond at the 5´splice site
The importance of being folded:
5´-site of splicing
site recognized by guanosine & site of the first attack
base-pairing
Specific primary, secondary, and tertiary structure is necessary for:
recognition of the guanosine binding site
recognition of the sites of splicing (attack)
guanosine binding site
RNA hairpin loop
3´-site of splicing
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
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
cyclic phosphate!
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
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 aminoacyltRNA 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, glucosamine6-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)
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