Nucleic Acids Research, 2005, Vol. 33, No. 5 1553

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Nucleic Acids Research, 2005, Vol. 33, No. 5
15531563
Characterization of the frameshift signal of Edr,
a mammalian example of programmed 1 ribosomal
frameshifting
Emily Manktelow, Kazuhiro Shigemoto1 and Ian
Brierley
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Background information
-1 Ribosomal frameshifting

• have been described in viruses, bacteria and
  eukaryotes
• the most widely used translational recoding
  mechanism of RNA viruses
• occurs during elongation phase of protein
  synthesis
• ribosome switches from zero reading frame to -1
• signals that promote frameshifting
• - slippery sequence
• - short spacer structure ? positions the
  pseudoknot on the ribosome when A and P sites are
  occupied by slippery sequence
• - 3-stimulatory RNA structure (typically
  an mRNA pseudoknot) ? induces ribosomes to pause

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Background information
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Background information
Mechanism
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Background information
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1. What was the biological question addressed?2.
What major claims do the authors make?3. What
are the major results that the authors
obtained?4. Did the experiments presented
justify the claims made?5. What major unanswered
questions were raised by this work?6. What
experiments need to be done to answer such
questions?
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Background information
Mouse Edr gene (PEG10)

• highly expressed during embryogenesis
• have different temporal patterns
• play crucial (but unknown) role in development
• the only eukaryotic gene which utilizes -1
  framshifting (Human Paraneoplastic Ma3 Gene)
• Two long ORFs (ORF1 and ORF2) were predicted
• ORF1 320 aa, Zn-binding motif
  (similar to Gag)
• ORF2 631aa, aspartyl protease
  catalytic site

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Background information
Ribosomal frameshifting analysis
Plasmid for in vitro transcription (SP6 RNA
Polymerase cap structure ? capped mRNA) RNA
extraction mRNA translation (Rabbit
reticulocyte lysate) SDS gel Autoradiography
detection Analysis of 35Smethionine
incorporation
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Results
Region downstream of NdeI is not important for
frameshifting
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Results
5 edge boundary is located between ?24 and ?64
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Results
Loop 1
Stem 2
Stem 1b
Stem 1a
Loop 2
New pseudoknot model was proven to be correct
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Results
sd C,G,A
dd
sd
sd C,G,A
New pseudoknot model was proven to be correct
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1. What was the biological question addressed?2.
What major claims do the authors make?3. What
are the major results that the authors
obtained?4. Did the experiments presented
justify the claims made?5. What major unanswered
questions were raised by this work?6. What
experiments need to be done to answer such
questions?
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Background information
Bypassing (hopping)

• similar to frameshifting, but slippage is more
  extensive
• tRNA pairs with mRNA at a non-overlapping codon
  
• identical codons flanking the nucleotides that
  were bypassed (take-off and landing site)
• the stop codon, the stem loop and the nascent
  peptide signal work synergistically to stimulate
  take-off
• 50 of ribosomes can bypass successfully

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Background information
Mechanism of bypassing
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1. What was the biological question addressed?2.
What major claims do the authors make?3. What
are the major results that the authors
obtained?4. Did the experiments presented
justify the claims made?5. What major unanswered
questions were raised by this work?6. What
experiments need to be done to answer such
questions?
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Background information
Bacteriophage T4 gene 60

• encodes topoisomerase subunit
• required for the tight association of p39 and
  p52 serves as a structural link, perhaps
  providing flexibility to the topoisomerase while
  holding the larger chains in juxtaposition
• relaxes positive or negative supercoils
• two open reading frames are separated by 50 nt
  (coding gap)

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Background information
Bypassing efficiency assay
Expression from GST-gene 60 fusion accounts for
40 of total protein synthesis ? easy detection
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Results
Does take off compete with termination???
no change in bypassing No direct competition of
bypassing with termination
ß-Gal
Growth at 41C Less binding of RF1 to
ribosomes Stop-hopping increases
Efficiency of bypassing is not defined by the
competition between termination and take-off at
normal conditions
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Results
Is local UAG context important for bypassing???
C ? A, G and U lowers the efficiency from 44 to
34, 39 and 33 Mutation reduced bypassing by
increased recognition
UAG context is important, but other factors are
involved
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Results
Is stability of the stem-loop influence
bypassing???
Efficiency was reduced 60, 30 and 30
Efficiency was reduced 2-fold
Stem-loop structure stimulates take-off
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Results
Is nascent peptide influence bypassing???
Inactivation of RF1 More affective bypassing
then in WT RF1-independent bypassing
Nascent peptide may contributes to the
RF1-independent bypassing
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Results
Does ribosomal protein L9 influence bypassing???
L9 influence the competition between take-off and
termination
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Results
Does ribosomal protein L9 influence bypassing???

Grey WT Red stem mutant Green nascent
peptide mutant Blue insertional mutant
L9 deficiency partially suppresses the defect in
the stem-loop
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1. What was the biological question addressed?2.
What major claims do the authors make?3. What
are the major results that the authors
obtained?4. Did the experiments presented
justify the claims made?5. What major unanswered
questions were raised by this work?6. What
experiments need to be done to answer such
questions?