Translational Bypass

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Translational Bypass
Bacteriophage T4 gene 60- component of T4 DNA
topoisomerase
50nt

• Other examples of bypass
• Hop of 2 codons in Bovine placental lactogen when
  overexpressed in E. coli.
• Bypass of 8 codons in the plaA gene of Prevotella
  loescheii by an unknown mechanism.

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tRNA2Gly- shuttles peptide over to landing site
Nascent peptide signal- reprogram ribosome
Stem-loop- reprogram ribosome for scanning
Stop codon- pause ribosome, stimulate take-off
GGA- specifies the endpoints of bypassing
Other players Ribosomal protein L9
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Only 50 of ribosomes productively bypass- Why do
the rest fail?
Termination competes with bypass (RF1 dependent)
Failure to land (RF1 independent)
How does bypassing occur?
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Major claims of the paper

• Bypassing is more efficient than termination
• Stop codon context is poorly recognized by RF1
• The stem-loop and nascent peptide function in
  distinct ways during bypassing

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Inactivation of RF1 increases UAG hopping but not
bypassing in gene 60
ß-galactosidase
ProtA-Cat
Activity of tsRF1
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Only overexpression of RF1 is able to decrease
bypassing efficiency in gene 60
Overexpressed RF1 from a ts promoter
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• In the wt strain (pGS1)
• 50 bypassing with wt levels RF1
• 50 bypassing with no RF1
• 44 bypassing with RF1 overexpressed
• Low level of read-through with amber
  suppressor tRNA

Full length bypassed fusion peptide
Read-through product
Termination product GST
RF1 wt level RF1 - tsRF1
overexpressed RF1
Amber suppressor tRNA
tRNA for GGA codon
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Of all possibilities for the stop codon context
UAGC allows for the most amount of
bypassing Bypassing is competing with
termination
44 34 39 33 Bypassing
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RF1 decodes UAA, UAG RF2 decodes UAA, UGA

• UAG bypasses better than UAA
• UGA is not affected by changes in RF1 because it
  only uses RF2

44 39 55 Bypassing
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Asp(GAU)-Asn(AAU)
Change from a negatively to positively charged AA
decreases bypassing in an RF1 dependent manner
44 34 bypassing
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Stable stem-loop region required for bypassing
pGS7- top 2 GC basepairs disrupted pGS8- stop
codon basepairs disrupted pGS16- stable tetraloop
changed
100 40 70 70
bypassing compared to wt
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Large insertion in the scanning region decreases
bypass efficiency
26nt insertion
100 50 bypassing compared
to wt
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Nascent polypeptide required for efficient
bypassing
Peptide mutation allows RF1 to compete with
bypassing Double mutant with SL has a
synergistic effect
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Trans effectors of bypassing
Ribosomes show decreased bypassing with the wt
gene60 without L9 or with mutant tRNA2Gly
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The stem and nascent peptide stimulate take-off
by separate mechanisms
Wt (pGS1) Stem mutant (pGS7) Nascent protein
mutant (pGS12) Long coding gap (pGS18)
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Conclusions
tRNA2Gly- shuttles peptide over to landing site
Stem-loop- reprogram ribosome for scanning,
requires L9
Nascent peptide signal- reprogram ribosome,
mutants suppress bypassing defects of mutant tRNA
Stop codon- pause ribosome, poor local context
for termination
GGA- specifies the endpoints of bypassing
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Unanswered Questions

• If UGA bypasses better than UAG then why isnt
  that the preferred stop codon in gene60?
• Maybe level of bypassing optimized to make the
  right amount of protein for virus survival
• Are there other factors that manipulate
  bypassing?
• What happens to the complexes that fail
  bypassing?
• How common is this mechanism? Did the
  bacteriophage capture it from a host?
• Is there a bypassing mechanism is eukaryotes?

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Edr (embryonal carcinoma differentiation
regulated)

• Human homologue PEG10
• present as a single copy in the genome
• Crucial role in mammalian development
• Organization similar to gag/pro in retroviruses

Clark, MB et al. 2007 JBC
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-1 Frameshift Requirements
GGGAAAC
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Claims of the paper

• Frameshifting occurs in vitro at 30 efficiency
  at the slippery site GGGAAAC
• The stimulatory RNA downstream of the slippery
  site is a pseudoknot
• This is similar to viral frameshift sites and
  does not represent a new cellular class of
  frameshift signals

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Edr frameshift occurs in vitro
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The region 3 to the slippery site required for
frameshifting is 80nt
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Revised model of the folding of the Edr
stimulatory RNA
H-type pseudoknot Similar to strategy of
retroviral IRESs
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Mutational analysis of the PK

• Slippery site sequence important for frameshift
  (m1, m2)
• Sequence of loop 2 not critical (m12)
• Bulge does not affect frameshifting (m13)

31 1.1 7.7 23.2 26.8 efficiency
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Structure but not sequence is important for the
stem regions of the PK
Why didnt they loop at Loop1?
31 12.2 4.9 25.6 1.7 2.1
24.6 1.8 2.0 16.5 efficiency
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Cleavage patterns U2 single stranded A
residues I single stranded regions CV1
double stranded and stacked bases Pb single
stranded region T1 single stranded G residues
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Cleavage patterns CL3 single stranded C
residues
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Summary of structural probing experiments
Alternative structure based on chemical probing
PEG10 sequence differences
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Conclusions

• Frameshifting occurs in vitro for the Edr mRNA at
  30 efficiency
• The slippery sequence is GGGAAAC
• The 3 stimulatory RNA is a H-type pseudoknot
  determined by mutagenesis analysis and structural
  probing assays

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Questions

• What is the most common conformation of the
  pseudoknot?
• Does the human homologue exhibit the same
  frameshifting? (Clark, MB et al. 2007 JBC)
• What is the in vivo efficiency?
• What happens if you disrupt this regulation in
  vivo?
• Are there other examples in Eukaryotes
• PRFdb is trying to answer that question

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-1 Frameshift Requirements
E. coli dnaX frameshift signal
or pseudoknot 3stimulatory structure
GGGAAAC