Ribozyme Structures and Mechanisms

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Ribozyme Structures and Mechanisms
Elizabeth A. Doherty Jennifer A. Doudna Annu.
Rev. Biochem. 2000. 69597615

• Reporter Chen Lu Qijia Wu

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Outline

• Introduction
• Ribozyme catalysis
• Self-splicing ribozymes
• RNase P
• Small self-cleaving ribozymes
• Other catalytic RNA

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Introduction

• Several naturally occurring classes of catalytic
  RNA have been identified to date.
• They catalyze cleavage or ligation of RNA or
  other biochemical reactions such as peptide-bond
  synthesis.
• We can compare ribozymes with enzymes. (Table.
  1.)
• Crystal structures of some ribozymes have been
  determined, providing detail of the tertiary
  folds of these RNA.

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Natural Ribozymes

• Large ribozymes
• Group I intron gt500 euk prok(rRNA, tRNA, mRNA)
• Group II intron gt100 euk prok(mRNA, tRNA,
  rRNA)
• RNase P gt100 euk prok
• Small ribozymes
• Hammerhead gt11 (plant viroids)
• Hairpin gt1 (satellite RNA)
• HDV gt2
• VS gt1 (Neurospora)
• Other ribozymes ribosome, spliceosome

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Table. 1 Comparison between ribozyme and enzyme
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Ribozyme Catalysis

• The acid-base catalysis Fig. 1a
• RNase A, hairpin, HDV.
• The two-metal ion catalysis Fig.1b
• Group I/II intron, RNase P, hammerhead.

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Fig. 1a The acid-base catalysis
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Fig. 1b The two-metal ion catalysis

• Some ribozymes catalyze phosphate chemistry
  through diffenrent mechanisms.

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How to test the model?

• Sulfer substitution
• Rescue by addition of Mn2.

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Self-splicing Ribozymes
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Group I Intron

• The first discoverd group I intron is the
  Tetrahymena thermophila group I intron in LSU
  rRNA.
• Nowadays group I introns are found in precursor
  rRNA, mRNA and tRNA.
• All group I introns have variable sequences but
  conserved advanced structures. (Fig. 2)
• Some introns contain ORFs encoding maturase.

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Fig. 2. The secondary structure model of group
I intron
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The mechanism of self-splicing

• The process is a two-step transesterification.(Fig
  . 3a)
• The reaction was assisted by 3 Mg2. (Fig. 3b)
• Self-splicing require the intron folding into an
  active structure. (Fig. 4)

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Fig. 3a the two-step transesterrification
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Fig. 3b The current model of first-step
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The structural model of Tt.LSU

• In 1990, a 3D-model of group I intron structure
  was constructed by comparative sequence analysis.
• The crystal structure of Tt.LSU had been solved
  at 5 Å resolution in 1998, and it proved the
  previous 3D model was right on the whole. (Fig.
  4)
• Some details will be discussed below.

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Fig. 4a The structure of Tt.LSU
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Fig. 4b The crystal structure of P5-P4-P6 and
P9-P7-P3-P8 domains (resolution is 2.8 Å)
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Some structure details

• There are many long-range interactions in the
  structure. (Fig. 4a)
• J8/7 is high conserved. It helps tightly pack the
  two main domains stack and forms a triple-helix
  with P1. (Fig. 5a)
• P5abc is not essential for group I intron
  function. But it not only stabilizes the core
  but also organize the detailed architecture of
  the core from a distance, preventing the
  accumulation of misfolded structures. (Fig. 5b)

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Fig. 5a Active site of the Tt.LSU
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Fig. 5bThe tertiary structure of P5-P4-P6 domain
and P5abc
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Group II Intron

• The self-splicing process of group II intron is
  different from group I intron, but the cleavage
  mechanism is similar. (Fig. 6)
• The secondary structure model of group II intron.
  (Fig. 7)
• We can compare group II intron with group I
  intron. (Table. 2)

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Fig. 6 The self-splicing of group I/II intron
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Fig. 7 Group II intron secondary structure model
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Table. 2 Comparison between group I/II intron
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RNase P

• RNase P is a key enzyme in the biosynthesis of
  tRNAs. It is an ribonucleoproteins (RNPs) that
  contain an RNA subunit essential for catalysis.
  (Fig. 8)
• The cleavage mechanism is similar to group I
  intron.
• The crystal structure of RNase P has been solved.
  (Fig. 9)

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Fig. 8. tRNA 5-end processing by RNase P
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Fig. 9. The structure of RNase P
Catalytic core
T-loop recognition
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Small self-cleaving RNAs

• Hammerhead Ribozyme Structure and catalysis
• Leadzyme Motif Structure and Catalysis
• Hepatitis Delta Virus Ribozymes Structure and
  Catalysis
• Hairpin Ribozyme Structure and Catalysis

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Hammerhead Ribozyme Structure and Catalysis

• The hammerhead ribozyme was initially discovered
  as a self-cleaving sequence within small RNA
  satellites of plant viruses which cleaves rolling
  circle replication products into genome-length
  units.

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Secondary Structure of a Minimal Hammerhead
Ribozyme

• Three Helices
• Highly conserved core of 15 bases
• Core bases non-complementary
• CUGA turn
• Splicing site C17

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Folding of Hammerhead Ribozyme

• The structure of domain 2 is formed in the first
  transition, from the nucleotides colored blue.
  Domain 1 forms in a second transition, occurring
  as the MgCl2 concentration rises above 1 mM, and
  involves the CUGA sequence colored magenta.

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Tertiary Structure of a Minimal Hammerhead
Ribozyme

• Three stems
• Arranged in a Y shape
• IIIII coaxial
• III form sharp angle
• Backbone distortions
• Magnesium binding sites

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Hammerhead Ribozyme Catalysis---Two-metal ion
model for ribozyme cleavage

• The metal ion in binding site 1 (Me1n)
  coordinates directly to the 2oxygen
• The resulting 2-alkoxide serves as the attacking
  nucleophile, displacing the 5-oxygen of the
  leaving nucleotide
• The metal ion in binding site 2 (Me2n) stabilize
  the leaving 5-oxygen

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Divalent cations debate

• There is no evidence for a metal ion contacting
  the 2-hydroxyl nucleophile.
• Recent studies show that the ribozyme can
  function in the complete absence of divalent
  cations at extremely high ionic strengths.

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Possible Mechanism

• 1Ground state geometry
• ?Transition
  state geometry

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2 sequence elements outside the hammerhead
ribozyme catalytic core may play some roles
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Hammerhead ribozyme summary

• - Scissile phosphate exposed to solvent
• - Inner sphere coordination of Mg2 at
• phosphate oxygen
• - Inactive ground state (observed in crystals)
• - High flexibility of catalytic center
• - Conformational change of scissile
• P-bond necessary

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Hairpin ribozyme Structure and Catalysis

• The hairpin ribozyme is found within RNA
  satellites of plant viruses, performing a
  reversible self-cleavage reaction to process the
  products of rolling circle genome replication.

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Hairpin Ribozyme Structure
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Formation of the Active Structure

• A sharp bend around the hinge between domains A
  and B enables the conserved regions to approach
  each other, buries the active core.
• Metal ions binding site at domain B.
• A10-C25, G11-A24 hydrogen bond in tertiary
  structure.

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Nucleotide Base Catalysis

• Changes in pH
• Substitution of sulfur for either of the two
  non-bridging oxygens
• Monovalent cations can also function

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Hairpin ribozyme summary

• Two domains act side by side
• Active site is buried between domains
• Tertiary contacts between domains gt
  flexibility
• No inner coordination of Mg2 at scissile
  phosphate
• Involvement of bases in acid / base catalysis ???

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Leadzyme Structure and Catalysis

• The leadzyme is a minimal catalytic motif derived
  from in vitro selection .
• Lead specific
• Two short watson-Crick duplexes
• An internal loop

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Leadzyme Structure
Pb

• Cleavage site CpG
• Pb binding site Gs8
• Contain no tertiary
• interaction.
• Unpaired region in the loop is important, perhaps
  because the stacking and hydrogen bonding between
  bases are important for the Gs8 positioning and
  Pb binding.

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Metal ions functions in structure and catalysis

• Two different conformations differring in their
  metal-binding properties
• A One leadzyme copy coordinates Mg2,but have no
  catalytic activity.
• B The other binds only Ba2 or Pb2. A single
  Ba2 ion coordinates the 2'-OH nucleophile in the
  core.

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HDV Ribozyme Structure and Catalysis

• satellite virus of HBV
• 1700 nt, 70 self-complementarity
• self cleaving of multimers
• 100 x faster than other small Ribozymes
• extremely stable
• active at 80C, 5 M urea
• substrate base paired only on 3-side
• a single nt at the 5-side is sufficient

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• - mainly watson-crick base pairs
• - GC base pairs are essential for stability
• - 5-G U wobble necessary for positioning
• - P1 sequence may vary freely

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• - no metal ions detected / only needed for 3
  structure stabilisation
• - 3 strand cross-overs and 2 G-C base pairs
  stabilize compact fold
• - 5-OH leaving group buried deeply between two
  domains
• - Cyt 75 extremely close to 5-OH leaving group
• - product and transition state structures are
  similar

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HDV Active Site

• C75 surrounded by negative charge from P-backbone

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stacking of 5-GU wobble fixes 5G-OH at the
active site close to C75
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Proposed mechanism for general acid-base
catalysis in the HDV

• Mg(OH) acting as a general base.
• C75 acting as a general acid.
• Interaction between N4 amino group of C75 and the
  pro-Rp oxygen of C22.