Structure of the 70s Ribosome Complexed with mRNA and tRNA

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Structure of the 70s Ribosome Complexed with mRNA
and tRNA

• wudi

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outline

• Crystal structure of the bacterial 70s ribosome
  refine to 2.8angstrom resolution.
• A metal ion stabilizes a kink in the mRNA that
  demarcates the boundary between A and P sites,
  preventing slippage of mRNA and stabilize the
  intersubunit interface.
• The interaction of E-site tRNA with the 50Ss
  subunit have both similarities and differences
  compared to those in the archael ribosome.

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previous work

• high resolution structures of the 50s and 30s
  ribosomal subunits were solved
• maps at 5.5 angstrom resolution of the whole 70s
  ribosome complexed with mRNA and tRNA.
• The structure of the E. coli ribosome was solved
  at 3.5 angstrom resolution.
• Electron cryomicroscopy yield detailed structures
  of various functional states of the ribosome

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Representative electron density in the 70S
structure
The acceptor end of P-site tRNA
23S RNA
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The large subunit protein L34 showing the
visibility of well-ordered side chains
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Mg ion is shown at the mRNA kink between the A
and P site as well as between the nucleotides
1400 and 1401 of h44 of 16S RNA
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P-site tRNA interactions with the ribosome
Overview of both RNA and protein interactions
with P-site tRNA. P-site tRNA interacts with many
ribosomal protein tails and 23S RNA. 16S RNA
bases interact with the anticodon stem, acting as
gate between the P and the E site
16S RNA
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Interaction of the anticodon loop of P-site tRNA
with mRNA and the 30S Subunit
The wobble base pair (C34-G3) is held in place
from two sides by 16S RNA base C1400 that stack
on the wobble base pair and by 16S RNA base G966
that stacks against the ribose of C34
16S RNA
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A-minor interactions of 16s RNA and P-site tRNA
Type I
A1339 of 16S RNA form type I A-minor interactions
with P-site tRNA base pairs G29-C41. The x
indicates that the distance is too long for the
hydrogen bond Type ll A-minor interaction of 16S
G1338 with P-site tRNA base pair G30-C42.
type II
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The gate between P and E site-tRNA
A1339 and G1338 of 16S RNA in the head of the 30S
interacts with the ASL of P-site tRNA on one
side, and 16s 790 at the tip of the 30S platform
contact the ASL on the other side,thus preventing
its movement into the E site
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P-site tRNA Interactions at the PTC
The CCA end of P-site tRNA in the PTC interacts
with conserved 23S RNA bases A2451 and the P
loop The 2-OH of A76 interacts with 23s A2451 C74
and C75 form Watson-Crick base pairs with P-loop
bases G2252 and G2251 .
23S RNA

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E-site interactions in the 30S
Anticodon of E-site tRNA does not interact with
the E-site codon of mRNA The author did not
observe any codon-anticodon interactions in the E
site
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E-site interactions in the 50S
A76 at the acceptor end of E-site tRNA
intercalates between G2421 and A2422 of 23S RNA
and makes hydrogen bond with the conserved
C2394. The 3-OH of A76 is surrounded by 23S RNA,
and the site could not accommodate an amino acid
on the tRNA.
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The sharp kink between the A- and P-site codons
clearly indicate the border between these two
sites and presumably is important for defining
the reading frame and preventing slippage of the
mRNA. In this 70s structure, this kink is
stabilized by a Mg ion, which makes interactions
with the phosphate oxygens of the third P-site
nucleotide and the first A-site nucleotide,
allowing them to come closer together.
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Overview of proteins

• The author report some significant differences
  from previous work
• A region of the 50S subunit was originally
  interpreted as corresponding to protein L31, but
  this region is now observed to ribosomal protein
  L28
• At the same time, unexplained density was found
  for a protein adjacent to protein L5. The authors
  suggest that this is protein L31 on the basis of
  the experiment.
• The author saw no evidence for L36. authors
  postulated that the protein is lost during the
  purification or is not a true ribosomal protein.

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Overview of the extensive intersubunit bridge
interactions
As described in the paper, there are so many
intersubunit bridge interactions in this
structure. RNA, side chains of protein and Mg
ions are all involved in intersubunit bridge
interaction.
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an example of an induced change in a bridge on
A-site tRNA binding
In bridge B2a, A1913 of H69 of 23S RNA flips out
of its loop toward 16S RNA bases A1492 and A1493
to form a hydrogen bond with A37 of A-site tRNA.
Two Mg ion also interact with A1913.
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A Mg mediated bridge
In bridge B2c, a Mg ion mediates the interaction
between the RNA backbones of h24 and h27 of 16S
RNA and H67 of 23S RNA.
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protein and ions in bridges
In bridge B5, Arg49 of L14 interacts directly
with the backbone of 1423 of 16S RNA Glu54 is
coordinated to a Mg ion that in turn interacts
with the backbones of nucleotide 1421 of 16S RNA
and 1950 and 1951 of 23S RNA
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protein and ions in bridges
Bridge B8 In the center, a Mg is coordinated to
the backbone oxygens of both Val115 and Ala118 of
L14 and to h14 of 16S RNA
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