Nucleic Acids Research, 2005, Vol. 33, No. 5 1553–1563

1. Nucleic Acids Research, 2005, Vol. 33, No. 5 1553–1563 Characterization of the frameshift signal of Edr, a mammalian example of programmed 1 ribosomal frameshifting Emily Manktelow, Kazuhiro Shigemoto1 and Ian Brierley*

2. 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

3. Background information

4. Background information Mechanism: 1 2 3

5. Background information

6. 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?

7. 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

8. 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 [35S]methionine incorporation

9. HindIII BamHII Results Region downstream of NdeI is not important for frameshifting

10. Results 5’ edge boundary is located between Δ24 and Δ64

11. Results Loop 1 Stem 2 Stem 1b Stem 1a Loop 2 New pseudoknot model was proven to be correct

12. Results dd sd sd C,G,A sd C,G,A New pseudoknot model was proven to be correct

13. 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?

15. 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

16. Background information Mechanism of bypassing

17. 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?

18. 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)

19. Background information Bypassing efficiency assay Expression from GST-gene 60 fusion accounts for ~40% of total protein synthesis  easy detection

20. Results Does take off compete with termination??? no change in bypassing No direct competition of bypassing with termination β-Gal Growth at 41°C 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

21. 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

22. 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

23. 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

24. Results Does ribosomal protein L9 influence bypassing??? L9 influence the competition between take-off and termination

25. 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

26. 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?