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Translational Bypass

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

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Translational Bypass

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

  2. 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

  3. RF1 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?

  4. 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

  5. Inactivation of RF1 increases UAG hopping but not bypassing in gene 60 ProtA-Cat β-galactosidase Activity of tsRF1

  6. Only overexpression of RF1 is able to decrease bypassing efficiency in gene 60 Overexpressed RF1 from a ts promoter

  7. 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

  8. 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

  9. 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

  10. Asp(GAU)-Asn(AAU) Change from a negatively to positively charged AA decreases bypassing in an RF1 dependent manner 44 34 %bypassing

  11. 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

  12. Large insertion in the scanning region decreases bypass efficiency 26nt insertion 100 50 %bypassing compared to wt

  13. Nascent polypeptide required for efficient bypassing Peptide mutation allows RF1 to compete with bypassing Double mutant with SL has a synergistic effect

  14. Trans effectors of bypassing Ribosomes show decreased bypassing with the wt gene60 without L9 or with mutant tRNA2Gly

  15. The stem and nascent peptide stimulate take-off by separate mechanisms Wt (pGS1) Stem mutant (pGS7) Nascent protein mutant (pGS12) Long coding gap (pGS18)

  16. 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

  17. Unanswered Questions • If UGA bypasses better than UAG then why isn’t 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?

  18. 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

  19. P A E X XXY YYZ XXX YYY Z 3’ stimulatory RNA structure stem-loop or pseudoknot Slippery sequence -1 Frameshift Requirements GGGAAAC

  20. 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

  21. Edr frameshift occurs in vitro

  22. The region 3’ to the slippery site required for frameshifting is 80nt

  23. Revised model of the folding of the Edr stimulatory RNA H-type pseudoknot Similar to strategy of retroviral IRESs

  24. Slippery site sequence important for frameshift (m1, m2) Sequence of loop 2 not critical (m12) Bulge does not affect frameshifting (m13) Mutational analysis of the PK 31 1.1 7.7 23.2 26.8% efficiency

  25. Structure but not sequence is important for the stem regions of the PK Why didn’t they loop at Loop1? 31 12.2 4.9 25.6 1.7 2.1 24.6 1.8 2.0 16.5% efficiency

  26. 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

  27. Cleavage patterns: CL3 – single stranded C residues

  28. Summary of structural probing experiments Alternative structure based on chemical probing PEG10 sequence differences

  29. 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

  30. 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

  31. -1 Frameshift Requirements E. coli dnaX frameshift signal or pseudoknot 3’stimulatory structure GGGAAAC

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