Ribozyme catalysis:not different,just worse

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Ribozyme catalysisnot different,just worse

• Jian Zhang
• 06.12.04

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Content

• Introduction
• RNA strand scission
• RNA splicing
• Peptide bond formation
• Conclusions

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Introduction

• The catalysts for biochemical reactions was
  important for the living systems.
• Protein enzymes dominate modern cell biology
• Discoveries of ribozymes fueled the suspicion
  that nucleic acids were key to the origin of
  biocatalysts

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• According to the RNA world hypothesis RNA once
  served as both the genetic material and the
  principal biocatalyst in living systems.
• As evolution goes on,enzymes with superior
  catalytic powers are required,so due to the
  transition to protein mediated catalysis.

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• Perhaps to sustain basic life forms on the early
  Earth.
• ribozymes are indeed capable of catalyzing
  chemical reactions and can provide rate
  enhancements.

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• Is ribozyme catalysis the same as or different
  than protein catalysis?
• At a chemical level,
• how different could RNA-based catalysis be from
  protein-based catalysis?

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• If we strip away the macromolecule from the
  catalyzed reaction , only a limited number of
  mechanisms are left.
• ????
• Proximation ????
• General acid-base catalysis
• Metalloenzyme electrostatic transition state
  stabilization and substrate orientation
• The overall goal
• stabilize the transition state of the reaction
  relative to the ground state.

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• This review highlights new insights into ribozyme
  catalysis as well as some of the many remaining
  challenges to understand detailed mechanisms of
  action of RNA enzymes.

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Table 1
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RNA strand scission

• Both protein enzymes and ribozymes catalyze
  phosphodiester bond scission by a variety of
  mechanisms demonstrating ribozymes potential
  surprisingly analogous to proteins
• But
• they act only at specific phosphodiester bonds by
  using base-pairing and other interactions to
  align the cleavage site within the RNA active
  site.

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• The hammerhead, HDV and hairpin ribozymes all
  catalyze site-specific self-cleavage during
  rolling circle replication of the viral or
  virusoid RNAs
• products 2',3'-cyclic phosphates and 5'-hydroxyl
  termini

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RNase A
nucleophilic attack of the 2' hydroxyl on the
adjacent phosphorous atom and produces products
with 2',3'-cyclic phosphate and 5'-hydroxyl
termini.
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The HDV ribozyme
Two proposed mechanisms for HDV ribozyme mediated
cleavage top, C75 acts as a general acid
bottom, C75 acts as a general base. Not all known
or proposed interactions are shown
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(c) Precursor and product states of the ribozyme
determined by X-ray crystallography.
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The hammerhead ribozyme

• Substrate orientation
• By treating one strand as the substrate and the
  other as the enzyme, multiple-turnover cleavage
  occurred
• Metalloenzyme ?
• The require for high (4 M) monovalent salt
  concentrations obviated a catalytic requirement
  for magnesium.
• The positions and functions of bound divalent
  metal ions have remained elusive.

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The HDV ribozyme

• Cleave intermediates generated during rolling
  circle replication of a human pathogen
• RNA might use general acid-base chemistry was
  first suggested by the structure of the
  self-cleaved form of the HDV ribozyme fig1b
• Proving whether and how general acid-base
  catalysis might work in the HDV and hairpin
  ribozymes remains an outstanding challenge.

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The hairpin ribozyme

• Cleave intermediates generated during rolling
  circle replication of a plant virus satellite RNA
• Whether a general base or acid is at work is
  murkier still for the hairpin ribozyme
• The lack of a requirement for divalent metal ions
  during hairpin ribozyme cleavage implies that it
  uses a metal ionindependent mechanism.

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• For the hammerhead and the HDV, structural
  information does not correspond to the
  information got from biochemical studies of the
  mechanisms of catalysis.
• Explaination the structures determined for these
  small ribozymes do not represent the structures
  that actually stabilize the transition states for
  the reactions
• perhaps because conformational changes must take
  place during the reaction cycle to attain the
  active states.

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RNA splicing

• Splicing involves the excision of an intervening
  sequence, or intron, from precursor transcripts
  to form a mature RNA
• Introns, occur widely within precursor
  transcripts of eukaryotic, and a few viral,
  messenger RNAs.

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• Splicing of these intervening sequences is
  catalyzed by the spliceosome
• But two different classes of autocatalytic
  introns are capable of self-excision.
• The group I class intron
• The group II class intron

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The group I class

• two-step transesterification mechanism initiated
  by an exogenous guanosine nucleoside or
  nucleotide.
• 1 the 3'hydroxyl of the bound guanosine
  substrate attacks the 5'-splice site phosphate
  and attaches to the 5' end of the intron
• 2 the 3' OH of the 5' exon attacks the
  phosphate at the 3'-splice junction,ligating the
  exons and excising the intron.

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• Analogous to protein enzymes, catalysis by group
  I introns requires divalent metal ions.three
  magnesium ions were proposed to contribute
  directly to catalysis

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Three metal ions

• (A) Stabilizes the developing Negative charge on
  the leaving group oxygen in the transition state
  and also destabilizes the bound substrate in the
  ground state.
• (B)Helps deprotonate the 3'oxygen of the G
  nucleophile
• (C)may aid both precise substrate positioning
  and, along with metal ion A,stabilization of the
  trigonal bipyramidal transition state

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Mechanism for PDE4
Two metal ions, a Mg2 and a Zn2, in the active
site of PDE4 ,coordinate either a hydroxide ion
or a water molecule,along with an aspartate, are
thought to orient the OH or H2O and promote its
attack on the phosphorus of the cyclic
nucleotide. The metals also serve to orient and
polarize the cyclic phosphate group. A histidine
is proposed to act as a general acid, protonating
the leaving group (3' oxygen).
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PDE4 active site as determined by X-ray
crystallography with substrate in orange and
oriented as in a.
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The group II class

• 1 the 2'OH of an internal adenosine within the
  intron serves as the initiating nucleophile
  cleaving the 5'splice site phosphodiester bond
  and forming a 2',5'linkage with the end of the
  intron.
• 2 the 3'OH of the 5'exon attacks the 3'splice
  junction phosphate, ligating the exons and
  releasing the branched lariat intron.

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Peptide bond formation

• ribozymes were largely supplanted by more
  efficient protein enzymes in the course of
  evolution.
• however, the catalyst still responsible for
  synthesizing nearly all proteins in cells is a
  ribozyme.

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• Except a few specialized peptides
• The vast majority of proteins are synthesized by
  the ribosome
• Biochemical evidence for a primary role of the
  RNA in this activity was bolstered by the
  discovery of an all-RNA active site in the
  peptidyl transferase center of the large
  ribosomal subunit

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• The ribosome uses two different substrate tRNAs
• the P site or peptidyl tRNA with the growing
  peptide chain attached by an ester linkage to its
  3' hydroxyl
• the A site or acceptor tRNA with a single amino
  acid esterified to its 3' hydroxyl
• the P-site tRNA substrate might contribute to
  catalysis of peptide bond formation.

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How ?
The amine on theA-site aminoacyl tRNA attacks the
carbonyl carbon of the P-site peptidyl tRNA to
produce an amide and an alcohol
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Challenges

• Challenges
• the presence of multiple genes encoding
  ribosomal RNAs in bacterial cells, coupled with
  the requirement of functional ribosomes for life
  ,so it is impossible to make pure populations of
  ribosomes with deleterious mutations in their
  rRNAs.

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Conclusion

• Nature has favored amino acidbased catalysts
  over RNA-based ones.
• Ribozymes are still extant and the lives of all
  organisms seem to depend on them.
• Like protein enzymes,ribozymes also use a lots of
  catalytic mechanisms ,perhaps ribozymes catalyze
  reactions in the same ways that proteins do.

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• Compare the details of RNA- and protein-based
  catalysis will help
• illuminate the reasons that ribozymes occupy
  indispensable niches in modern biology
• provide insight into the catalytic roles RNA may
  play within complex ribonucleoproteins

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Thank you