Translational Recoding

1. Translational Recoding David Bedwell Post-Transcriptional Regulatory Mechanisms Advanced Course MIC759 Sept. 5, 2006

2. Myths in Modern Molecular Biology • The “universal genetic code” is universal. • The genetic code is unambiguous. • All DNA encodes the information to make proteins with 20 amino acids. • The “central dogma of molecular biology” describes the only flow of biological information. • Eukaryotic translation initiation only occurs in a cap-dependent manner (a la Kozak). • Recoding mechanisms frequently represent exceptions to established dogma and highlight functional features of underlying mechanisms.

3. Translation Start Site Selection in Eukaryotes Ternary complex 43S pre-initiation complex eIF4F cap-binding complex Gebauer & Hentze Nature Reviews Molecular Cell Biology 5, 827-835 (2004)

4. Translation Start Site Selection in Prokaryotes mRNA 16S rRNA The efficiency of translation initiation is determined by: 1) Complementarity of the Shine-Dalgarno (SD) sequence with the 3´ end of 16S rRNA. 2) Distance between the SD sequence and the start codon (a 7 base spacer is optimal). Voet and Voet, Biochemistry, 2nd Ed., Figure 30-42

5. e) 50S EF2:GDP AA-tRNA AA-tRNA:EF1A:GTP d) 30S 50S EF1A:GTP EF1B a) 50S 30S GTP 30S EF1A:EF1B c) EF2:GTP 50S b) GDP EF1A:GDP 50S EF1B 30S 30S Translation Elongation Translocation & GTP hydrolysis tRNA selection Hybrid state GTP hydrolysis & proofreading Peptide bond formation EF1A = EF-Tu EF1B = EF-Ts EF2 = EF-G Merrick & Nyborg, The Protein Synthesis Elongation Cycle, In Translational Control of Gene Expression (2000), CSHL Press, NY

6. Hybrid States Model for Translocation Ramakrishnan, Cell 108: 557-572 (2002)

7. tRNA Selection During Elongation Ramakrishnan, Cell 108: 557-572 (2002)

8. A Key Determinant of Ribosome Fidelity is Helix 44 of the Small Subunit rRNA Helix 44 “Decoding Site” Ramakrishnan, Cell 108: 557-572 (2002) Yusupov et al., Science 292: 883-896 (2002)

9. (Helix 34) (Helix 18) (Helix 44) (Helix 44) Critical Nature of Residues A1492 and A1493 of Helix 44 in Translational Fidelity Ogle et al. (2002) Science 292:897-902.

10. Comparison of eukaryotic and prokaryotic termination factors

11. Prokaryotic Translation termination Zavialov et al., Cell 107: 115-124 (2001)

12. Eukaryotic Translation Termination Alkalaeva et al., Cell 125: 1125-1136 (2006)

13. Recoding mechanisms include: • Ribosomal frameshifting • +1 frameshifting • -1 frameshifting • Ribosome hopping • Stop codon readthrough • Incorporation of unusual amino acids at stop codons • selenocysteine • pyrrolysine

14. RF2 expression in bacteria is subject to feedback control by its own activity • RF-2 recognizes UAA and UGA, while RF-1 recognizes UAA and UAG stop codons. • The RF-2 ORF contains an in-frame UGA stop codon and a good SD sequence 3 nucleotides upstream of the frameshift site (5´-CUU UGA C-3´). • When the RF-2 level is low, the ribosome pauses when a UGA codon is located in the A site. tRNAleu in the P site then slips from the CUU codon to the UUU codon. • Frameshifting is enhanced by the presence of SD-like element 3 nucleotides upstream (suggests a push-forward mechanism?). • In this way, more RF-2 is made when there is not enough to rapidly terminate translation at the UGA stop codon.

15. * * * * * E. coli S30 RRL Donley & Tate, Proc Biol Sci 244: 207-210 (1991) Namy et al., Mol Cell 13: 157-1698 (2004) +1 Frameshifting Required for E. coli RF-2 Synthesis

16. Cellular Polyamine Levels Control Antizyme 1 Synthesis • Polyamines like spermine and spermidine are found in both prokaryotes and eukaryotes, where they stabilize membranes, ribosomes, DNA, viruses, etc. • Cellular polyamine levels are regulated by antizyme 1 in eukaryotes. • High polyamine levels stimulate the synthesis of antizyme 1. • Antizyme 1 then binds to ornithine decarboxylase (ODC) and triggers its degradation by the 26S proteosome (in an unusual ubiquitin-independent manner). • Since ODC catalyzes the 1st step in polyamine synthesis, its degradation leads to reduced polyamine synthesis. • Reduced polyamine levels reduce antizyme 1 expression. • Antizyme expression controlled by +1 frameshifting mechanism induced by high polyamine levels. • Required elements include polyamines, a “shifty stop” slippery sequence (5´-UCC UGA U-3´) at the frameshift site, and a pseudoknot just 3´ of the slippery sequence.

17. +1 Frameshifting in Antizyme Synthesis Pseudoknot Structure “shifty stop” slippery sequence (5´-UCC UGA-3´) Namy et al., Mol Cell 13: 157-1698 (2004)

18. +1 Frameshifting in the yeast EST3 gene • EST3 encodes a subunit of telomerase with an internal programmed +1 frameshift site between ORF1 (93 AAs) and ORF 2 (92 AAs) in S. cerevisiae and also many other yeast species. • The frameshift site has the slippery sequence 5´-CUU AGU U-3´. • AGU is encoded by a low abundance tRNA (sometimes referred to as a “hungry codon”), which frequently induces a ribosomal pause. • During pausing, the tRNAleu in the P site can undergo +1 slippage to the overlapping UUA codon. • May be other required elements, but not known yet.

19. EST3 +1 frameshifting is conserved in many yeast species Conservation of this slippery site among many related yeast species over millions of years of evolution suggests frameshifting may play some important role in telomere maintenance. Namy et al., Mol Cell 13: 157-1698 (2004)

20. -1 frameshifting is common in retroviruses (including HIV) and other viruses Model of Beet Western Yellow Virus (BWYV) -1 frameshift. Bases in red are conserved in all known luteoviruses. Frameshifting requires a 7 nucleotide slippery site and a downstream pseudoknot. Alam et al., Proc Natl Acad Sci USA 96: 14177-14179 (1999)

21. Retroviral -1 frameshifting • Retroviral -1 frameshifting between the Gag and Pol reading frames occurs about 5-10% of the time. • Gag includes the structural proteins matrix, capsid, and nucleocapsid. • Pol encodes the reverse transcriptase, endonuclease/integrase, and the viral protease. • Mutants that eliminated the -1 frameshift or made the Gag and Pol ORFs in-frame both eliminated the production of infectious virus. • Thus, the ratio of Gag to Gag-Pol conferred by frameshifting is critical for the viral life cycle.

22. While rare in cellular genes, -1 frameshifting occurs in the E. coli DnaX gene • The E. coli DnaX gene encodes two subunits of DNA Polymerase III: the  subunit is the product of normal translation, while the  subunit is derived by -1 frameshifting. • Frameshifting occurs at the slippery sequence 5´-A AAA AAG-3´ by simultaneous slippage of both the P and A site tRNAlys species in the -1 direction. • Frameshifting requires an SD-like element 10 nucleotides upstream of the slippery sequence and a stem-loop structure 5 nucleotides downstream of the frameshift element. • The extra distance to the SD element may enhance the realignment (suggesting a pull-back mechanism).

23. -1 Frameshifting in the E. coli DnaX gene DNA Pol III 10 nucleotides Namy et al., Mol Cell 13: 157-1698 (2004)

24. Ribosome Hopping • Bacteriophage T4 gene 60 bypassing requires: • - matching GGA codons flanking an optimally sized 50 nt coding gap • - a stop codon • - a stem loop structure • a nascent peptide signal. • In the current model, peptidyl-tRNA2Gly detaches from the take-off site GGA, scans the mRNA as it slides through the P-site, then pairs with the landing-site GGA. Nearly all ribosomes initiate scanning, and ~50% resume translation in the second ORF. Herr et al., EMBO J 19: 2671-2680 (2000)

25. Incorporation of selenocysteine, the 21st amino acid, occurs at in-frame UGA codons • Whenever a stop codon enters the ribosomal A site, a competition occurs between the class I release factor(s) and near-cognate tRNAs (that can base pair at 2 of the 3 nucleotides of the stop codon). • The release factor normally wins this competition 99.9% of the time, but this efficiency can be reduced by the sequence context around the stop codon, the relative level of the release factor, and the presence of downstream elements that can stimulate suppression. • Selenocysteine incorporation requires a selenocysteine insertion element (SECIS). • In eubacteria, the specialized translation elongation factor SelB binds both the SECIS just downstream of the SECIS and tRNA(ser)sec. • In eukaryotes, the SECIS is located in the 3´-UTR of the mRNA. Association of mSelB (also known as eEFsec) to the SECIS element requires the adaptor protein SBP2.

26. Mechanism of selenocysteine incorporation in prokaryotes and eukaryotes • The translation elongation factor SelB (or mSelB) that delivers tRNA(ser)secUCAto the A site is functionally analogous eEF1A (but no known GTPase activity). • One or two SECIS elements in the 3´-UTR of a eukaryotic mRNA can mediate selenocysteine incorporation at many UGA codons in the mRNA. • For example, expression of selenoprotein P in zebrafish requires the reassignment of 17 UGA codons (!). This suggests that selenocysteine incorporation can be very efficient. Namy et al., Mol Cell 13: 157-1698 (2004)

27. Similar SECIS elements mediate selenocysteine incorporation in prokaryotes and eukaryotes, but their location differ Consensus Hatfield & Gladyshev, Mol Cell Biol 22: 3565-3576 (2002) Namy et al., Mol Cell 13: 157-1698 (2004)

28. Examples of selenocysteine-containing proteins in animals Many selenoproteins are found in animal cells. Consistent with their frequent occurrence, selenoproteins are essential for mammalian development, since a tRNA(ser)sec knockout mouse is embryonic lethal. Hatfield & Gladyshev, Mol Cell Biol 22: 3565-3576 (2002)

29. Pyrrolysine, the 22 AA, is encoded by UAG codons in methanogenic Archaebacteria Pyrrolysine is an amide-linked 4-substituted pyrroline-5-carboxylate lysine derivative. It is found only in methanogenic Archaebacteria. It occurs in proteins that assist with the utilization of methanogenic substrates like trimethylamines. Each substrate requires activation by a methyltransferase to generate methane. All known methylamine methyltransferase genes contain pyrrolysine encoded at UAG codons.

30. Pyrrolysine, the 22 AA, is encoded by UAG codons in methanogenic Archaebacteria Little is currently known about the mechanism of pyrrolysine insertion at UAG codons. However, potential pyrrolysine insertion (PYLIS) elements can be found 5-6 bases downstream of the sites of insertion. Namy et al., Mol Cell 13: 157-1698 (2004)

31. Programmed stop codon readthrough in viral genes Beier & Grimm, Nucl. Acids Res 29: 4767-4782 (2001)

32. Programmed stop codon readthrough in MuLV Beier & Grimm, Nucl. Acids Res 29: 4767-4782 (2001)

33. Programmed stop codon readthrough in MuLV requires a downstream pseudoknot Beier & Grimm, Nucl. Acids Res 29: 4767-4782 (2001)

34. (Helix 34) (Helix 18) Recall AA-tRNA proofreading during tRNA selection. (Helix 44) (Helix 44) Ogle et al. (2002) Science 292:897-902. Pharmacological suppression of stop codons • Certain drugs can bind to the ribosome and reduce the ability • Aminoglycosides • PTC124 • May allow the treatment of a broad array of genetic diseases caused by premature stop mutations • What is the mechanism of aminoglycoside suppression?

35. Paromomycin bound Unbound Aminoglycosides Bind Helix 44 and Reduce Ribosomal Fidelity During Translation Aminoglycoside binding to Helix 44 leads to reduced elongation fidelity (misreading) and less efficient translation termination (readthrough). Yoshizawa et al, EMBO J. 17: 6437-6448 (1998); Ogle et al, Science 292:897-902 (2001)

36. Reassignment of the Genetic Code: The Genetic Code and its Variants Blue letters: changes in mitochondrial lineages. Bold letters: changes in nuclear lineages. Blue boxes: codons that have changed only in mitochondria. Green boxes: codons that have changed both in mitochondrial and in nuclear lineages. No codons have changed in nuclear lineages only. (Standard one-letter codes are used for reassigned amino acids; ?, unassigned. Subscripts give number of changes; the minus sign indicates reverse change.) Knight et al., Nature Rev Genetics 2: 49-58 (2001)

37. Phylogenetic Tree of eRF1 Molecules and Associated Stop Codon Usage Stop codon reassignment in cilates: UGA-only ciliates arose independently at least 3 times: in (Stylonichia+Oxytricha), in Loxodes, and in (Tetrahymena+ Paramecium). UAA/UAG-specific ciliates arose at least twice independently, in Euplotes and in Blepharisma. Kim et al., Gene 346: 277 (2005)

38. Euplotes: UAA/UAG only Extremely High Rates of +1 Frameshifting Occur in Euplotes Species • Euplotes species use UAA and UAG as stop codons, and have recoded UGA as a cysteine codon. • Most organisms have an extremely low incidence of translational frameshifting (e.g., frameshifting occurs in only 3 out of 6000 genes in yeast). • 8 out of 90 Euplotes genes sequenced to date have in-frame +1 frameshift sites with the similar “shifty stop” motif 5´-AAA UAA A-3´ (all but one uses the UAA stop codon). • Suggests frameshifting linked to stop codon reassignment. Klobutcher and Farabaugh, Cell 111:763-6 (2002) Klobutcher, Euk Cell 4: 2098-2105 (2005)