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Ribozyme Structures and Mechanisms

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Title: Ribozyme Structures and Mechanisms


1
Ribozyme Structures and Mechanisms
Elizabeth A. Doherty Jennifer A. Doudna Annu.
Rev. Biochem. 2000. 69597615
  • Reporter Chen Lu Qijia Wu

2
Outline
  • Introduction
  • Ribozyme catalysis
  • Self-splicing ribozymes
  • RNase P
  • Small self-cleaving ribozymes
  • Other catalytic RNA

3
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.

4
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

5
Table. 1 Comparison between ribozyme and enzyme
6
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.

7
Fig. 1a The acid-base catalysis
8
Fig. 1b The two-metal ion catalysis
  • Some ribozymes catalyze phosphate chemistry
    through diffenrent mechanisms.

9
How to test the model?
  • Sulfer substitution
  • Rescue by addition of Mn2.

10
Self-splicing Ribozymes
11
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.

12
Fig. 2. The secondary structure model of group
I intron
13
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)

14
Fig. 3a the two-step transesterrification
15
Fig. 3b The current model of first-step
16
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.

17
Fig. 4a The structure of Tt.LSU
18
Fig. 4b The crystal structure of P5-P4-P6 and
P9-P7-P3-P8 domains (resolution is 2.8 Å)
19
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)

20
Fig. 5a Active site of the Tt.LSU
21
Fig. 5bThe tertiary structure of P5-P4-P6 domain
and P5abc
22
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)

23
Fig. 6 The self-splicing of group I/II intron
24
Fig. 7 Group II intron secondary structure model
25
Table. 2 Comparison between group I/II intron
26
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)

27
Fig. 8. tRNA 5-end processing by RNase P
28
Fig. 9. The structure of RNase P
Catalytic core
T-loop recognition
29
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

30
(No Transcript)
31
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.

32
Secondary Structure of a Minimal Hammerhead
Ribozyme
  • Three Helices
  • Highly conserved core of 15 bases
  • Core bases non-complementary
  • CUGA turn
  • Splicing site C17

33
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.

34
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

35
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

36
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.

37
Possible Mechanism
  • 1Ground state geometry
  • ?Transition
    state geometry

38
2 sequence elements outside the hammerhead
ribozyme catalytic core may play some roles
39
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

40
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.

41
Hairpin Ribozyme Structure
42
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.

43
Nucleotide Base Catalysis
  • Changes in pH
  • Substitution of sulfur for either of the two
    non-bridging oxygens
  • Monovalent cations can also function

44
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 ???

45
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

46
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.

47
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.

48
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

49
  • - mainly watson-crick base pairs
  • - GC base pairs are essential for stability
  • - 5-G U wobble necessary for positioning
  • - P1 sequence may vary freely

50
  • - 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

51
HDV Active Site
  • C75 surrounded by negative charge from P-backbone

52
stacking of 5-GU wobble fixes 5G-OH at the
active site close to C75
53
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.
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