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RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal

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... the active site, displacing the aG and positioning the 3'exon for exon ligation. ... Exon ligation and intron release are accomplished by the O3' of the 5'exon ... – PowerPoint PPT presentation

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Title: RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal


1
RNA splicing group I intron crystal
structures reveal the basis of splice site
selection and metal ion catalysis Mary
R Stahley and Scott A Strobel Current
Opinion in Structural Biology 2006, 1618

Zhang Xiaorong
2
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  • ??1/3??????????????
  • (1)??? ????????????????? ?Fe 3 Fe 2 Cu 2 Zn
    2 Mn 2 Co 2
  • (2)????? ?????????????,?????,????????????????????
    ?2??Na 1 K 1 Ca 2 Mg 2

3
  • The group I intron has served as a model for
    RNA catalysis since its discovery 25 years ago.
    Four recently determined high-resolution crystal
    structures complement extensive biochemical
    studies on this system. Structures of the
    Azoarcus, Tetrahymena and bacteriophage Twort
    group I introns mimic different states of the
    splicing or ribozyme reaction pathway and provide
    information on splice site selection and metal
    ion catalysis.

4
  • The 5splice site is selected by formation of a
    conserved GU wobble pair between the 5exon
    terminus and the intron.
  • The 3splice site is identified through
    stacking of three base triples, in which the
    middle triple contains the conserved terminal
    nucleotide of the intron, OG.

5
  • The structures support a two-metal-ion
    mechanism for group I intron splicing that might
    have corollaries to group II intron and pre-mRNA
    splicing by the spliceosome.
  • The two-metal-ion mechanism for catalytic RNA
    was first elucidated by Steitz and Steitz in
    1993 which published on PNAS

6
Since a lot of protein phosphoryltransfer enzymes
used two-metal-ion mechanism and they does not
require that protein side chains of the enzyme
participate directly in the chemistry of the
reaction. Thus, the mechanism can be easily
accommodated by an enzyme composed of RNA.
By Steitz and Steitz in 1993
7
contents
  • Introduction
  • Capturing reaction states
  • Splice site selection
  • Active site metals
  • Metal ion catalysis in RNA splicing
  • conclusions

8
Introduction
  • RNA enzymes, or ribozymes, catalyze a variety
    of reactions including protein synthesis,
    self-splicing, self cleavage and tRNA 5end
    formation . RNAs active role in the cell was
    first illuminated with the self-splicing activity
    of the Tetrahymena thermophila pre-rRNA .
  • But only recently have these efforts yielded
    molecular resolution structural information that
    includes the complete
  • active site.

9
  • This review discusses the four group I intron
    crystalstructures published in 2004 and 2005
  • two group I intronexon complexes from
    pre-tRNAIle in Azoarcussp. BH72 , a Tetrahymena
    pre-rRNA apo enzyme and a bacteriophage Twort
    ribozyme-product complex from a pre-mRNA .

10
  • The focus of this review is the structural
    basis of splice site selection and metal ion
    catalysis as observed in these crystal
    structures. The metal binding pocket of the group
    I intron active site will be compared to RNA,
    protein and ribonucleoprotein complexes that
    perform similar phosphoryl transfer reactions.

11
Capturing reaction states
  • Group I introns self-splice via two
    consecutive phosphoryl transfer reactions In the
    presence of Mg2, a bound exogenous G(aG)
    initiates splicing with attack of its O3 on the
    5splice site. A conformational change then
    brings the terminal nucleotide of the intron,OG,
    into the active site, displacing the aG and
    positioning the 3exon for exon ligation.

12
  • aG attack its O3 on the 5splice site
  • Conformation change displace aG with OG
  • Exon ligation and intron release are
    accomplished by the O3 of the 5exon attacking
    the 3-splice site.

13
  • In this RNA, the 3 and 5ends of the intron
    are removed ,and an aG cleaves, in trans, an
    oligonucleotide at the 5splice site. The
    reaction is analogous to the first step of
    splicing

14
  • In order to mimicking different states of the
    reaction pathway, crystal of different intron
    subclasses and different reaction states are
    represented. In each case ,modifications or
    deletions were made to promote crystallization
    and capture a particular reaction intermediate.
  • (1) The first Azoarcus structure has four
    2deoxy substitutions in the active site,
    including a substitution at the last nucleotide
    of the 5exon (U-1). Mimic pre-1S
  • (2)The second Azoarcus structure restores three
    of the four riboses, including OG, but retains a
    single U-1 substitution. Mimic pre-2S

15
  • (3)The Tetrahymena group I intron structure (at
    3.8 A resolution) is comparable to the apo form
    that lacks both exons and the complementary
  • sequence in the intron to which they bind
    (the internal guide sequence, IGS), but it does
    include the OG . Mimic the 1st step of splicing
  • (4)The bacteriophage Twort group I intron 50-exon
    complex (at3.6 A resolution) is similar to the
    product of the ribozyme reaction, although it
    also has the OG. The Twort structure omits the
    scissile phosphate, the 3exon and the portion of
    the IGS complementary to the 3exon.mimic the
    product.

16
Azoarcus deoxy-wG pre-2S structure
17
Mimic the first step
Tetrahymena apo enzyme structure
18
Mimic product
Structure of Twort group I intron with P1 helix
19
OG
Group Iintron secondary structure with G binding
site
Superposition of three intron ribbon structure
Azoarcus (grey), Tetrahymena (gold) and Twort
(green)
20
  • Despite these differences in reaction state
    and subclass, the four structures fold into a
    single 3D structure within the conserved paired
    and joining regions . Two key elements of the
    group I intron self-splicing reaction can now be
    structurally examined
  • How does the intron select the 5and 3splice
    sites?
  • How does the RNA catalyze the splicing
    reactions?

21
Splice site selection
  • Accurate splicing requires selecting two
    specific phosphates among hundreds for cleavage
    and ligation reactions. How group I introns
    achieve this selection using secondary and
    tertiary interactions is visualized in these
    structures.

22
Group I intron 5splice site selection is based
on recognition of a conserved wobble pair in the
substrate helix formed between the terminal
nucleotide of the 5exon,U-1, and a conserved G
within the introns IGS .The substrate helix
makes tertiary contacts to two conserved regions
within the active site, J4/5 and J8/7.
  • 5Splice site selection. The GU wobble pair
    is recognized by stacked As in the J4/5 wobble
    receptor.
  • Coordinates from Azoarcus ribo-OG structure.
    U-1 o2 is modeled (grey).

23
The substrate helix makes tertiary contacts to
two conserved regions within the active site,
J4/5 and J8/7.
24
3Splice site selection is achieved through
specific binding of the intron s conserved
terminal nucleotide, OG, which forms a triple
with a GC pair in the P7 helix . All four group
I intron structures contain the OG and all
demonstrate how the P7 region folds to form the G
binding site The OG triple is stacked between two
additional P7 triples, which suggests that the
introns terminal nucleotide is selected through
hydrogen bonding and stacking interactions.
25
  • 3Splice site selection. Coordinates from
    Tetrahymena structure.molecule D. O G forms a
    GCG triple at the center of the G binding site.
    Two additional triples stack above and below to
    complete the G binding through stacking
    interactions

G binding to the group I intron occurs in two
stages an initial misaligned binding, followed
by a conformational change placing the G in an
active orientation .
26
Twort
Tetrahymena
Azoarcus
27
a, Side view of the active site oriented to
emphasize the stacking interactions that mediate
QdG206 binding. The two active-site metal ions
are shown as black spheres. b, Top-down view of
the base triple between the unpaired A129 and the
closing G128C178 base pair in the P7 helix. c,
Top-down view of the base triple between OdG206
and the G130C177 base pair
28
Active site metals
  • Group I intron splicing requires divalent
    metal ions for proper folding and catalysis . Six
    active site oxygens have been biochemically
    implicated for direct metal ion coordination
    through metal specificity switch experiments
    These assays substitute oxygens with soft
    atoms, such as sulfur or nitrogen, which are less
    able to coordinate Mg 2, a hard metal. If the
    addition of a soft cation (such as Mn 2 or Cd
    2) rescues interference due to the
    substitution,the oxygen is considered a metal
    ligand.

29
Group I intron splicing requires divalent
metal ions for proper folding and catalysis. Six
active site oxygens have been biochemically
implicated for direct metal ion coordination
30
  • Mg2 coordination to substrate oxygens
    activates the nucleophile(M1), stabilizes the
    scissile phosphate and stabilizes the developing
    charge on the leaving group(M2).
  • The ribo-OG Azoarcus group I intron crystal
  • structure includes all these ligands . This
    structure shows two Mg2 ions bound in the active
    site with all biochemically defined metal ligands
    satisfied, including all those in a previously
    proposed three-metal-ion model

31
5exon(U1) 3exon OG Mg2 ions K
This structure shows two Mg2 ions bound in the
active site with all biochemicallydefined metal
ligands satisfied, including all those in a
previously proposed three-metal-ion model.
32
M1,which coordinates the nucleophile of the
second step, is equivalent to the biochemically
termed MA. M2 has the coordination of MB and MC
combined, as it includescoordination to both the
O3 and O2 of the OG
metal ion interactions with three substrate
atoms, the 3-oxygen of the oligonucleotide
substrate and the 3- and 2-moieties of the
guanosine nucleophile, are mediated by three
distinct metal ions.(Herschlag)
33
This structure shows two Mg2 ions (referred to
as M1 and M2) bound in the active site with all
biochemically defined metal ligands satisfied,
including all those in a previously proposed
three-metal-ion model. (ribo-OG Azoarcus)
34
The 2-OH is a metal ion binding ligand. Removal
of the 2-OH at the terminal nucleotide, causes a
structural rearrangement that switches the metal
specificity from a Mg2 in the ribo-wG structure
to a K bound in the deoxy-wG structure
35
Metal ion catalysis in RNA splicing
  • Steitz and Steitz proposed that group I
    introns , group II introns and the spliceosome
    catalyze phosphoryl transfer reactions with the
    same two-metal-ion mechanism seen in protein
    polymerases and phosphatases. The ribo-OG
    Azoarcus group I intron crystal structure
    provides the first structural evidence for this
    model.

36
(a) Group I intron Azoarcus ribo-wG structure
and (b) T7 DNA polymerase.
The RNA intron uses its phosphate backbone to
form an active site metal binding pocket in the
same way that proteinaceous polymerases use
carboxylates of aspartate side chains.
37
an open octahedral coordination position on the
leaving group stabilizing metal M2 for a
metal-coordinated water Although this water was
not visible at the resolution of the ribo-OG
structure, an intriguing possibility is that this
water could protonate the O3 OG leaving group
38
  • Group II introns and the spliceosome might
    catalyze exon ligation using the same
    two-metal-ion architecture observed in the group
    I intron. Metal specificity switch experiments
    have been performed on active site oxygens in all
    three systems and the interference and metal
    rescue data indicate analogous metal ion
    coordination.

39
Biochemically implicated catalytic metal ion
ligands in RNA-based splicing exon ligation.
Active site oxygens that show interference
(sulfur or amino substitution) followed by soft
metal rescue are shown in red .Oxygens that show
only interference are shown in orange .The ?
indicates a position that has not been S or N
substituted 41. Note that double sulfur
substitutions required for the scissile
phosphates pro-RP oxygen metal specificity
switch in the group I intron have not been
performed in the group II intron and spliceosome.
40
Conclusions
  • The four group I intron structures described
    here have demonstrated the structural basis for
    splice site selection and metal ion catalysis. A
    two-metal-ion mechanism of group I intron
    splicing is supported by these structures,
    consistent with a large body of biochemical data.
    Armed with this picture of RNA-catalyzed splicing
    and biochemical analysis, similarities between
    metal ion catalysis of exon ligation by the group
    I intron, group II intron and
  • spliceosome are beginning to emerge.

41
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