1. Translation Tom Kristensen, tom.kristensen@ibv.uio.no

2. Fig.15.1 Three possible reading frames of the E. coli trp leader sequence Start codon: AUG, in bacteria also GUG and even UUG Stop codons: UAG, UGA and UAA

3. Fig.15.2 Structure of messenger-RNA from prokaryotes and eukaryotes Kozak sequence. Many eukaryotic RNAs lack these bases, but their presence increase the efficiency of translation Many prokaryotic RNAs contain two or more ORFs and encode more than one protein. We call them polycistronic RNAs. A polycistronic RNA often encode proteins that perform related functions, such as different steps in the biosynthesis of an amino acid or nucleotide. The ORFs will not always have their own RBS (ribosome binding site). Sometimes the ORFs overlap (most often as the sequence (5’- AUGA), making it possible for the ribosome to start a new translation without letting go of the mRNA. Eukaryotic RNAs only contain one ORF. No ribosome binding site is present in the sequence. Instead, the 5’ cap is used to recruit the ribosome. After binding to the cap, the ribosome scans the mRNA until an AUG is encountered. The poly-A tail enhances the level of translation of the mRNA by promoting efficient recycling of ribosomes.

4. Fig.15.3 and 4 The secondary structure of tRNA: the cloverleaf structure Bracketed by purine in the 3’ and uracil in the 5’ end (due to the presence of dihydrouridine) (due to the presence of pseudouridine) This end is added separately by a specific enzyme. The amino acid is bound to the terminal A

5. Fig.15.5 From the cloverleaf to the actual 3D structure Note: This figure only intends to show the actual structure is related to the cloverleaf representation. It does not pretend to show the actual folding pathway!

6. Fig.15.6 Coupling an amino acid to a tRNA molecule Two steps: (a)Formation of an aminoacyladenylate, a mixed acid anhydride that remains closely associated with the enzyme (b)Transfer of the aminoacyl residue to tRNA

7. Table 15-1 Class I enzymes add the amino acid to the 2’-OH group and are typically monomeric, while class II enzymes add the amino acid to the 3’-OH group of the tRNA and are typically dimeric or tetrameric. Most organisms have 20 aminoacyl-tRNA synthetases, one for each amino acid. Some bacteria lack a Gln-tRNA synthetase and instead use the Glu-tRNA synthetase to charge tRNA Gln, whereafter Glu-tRNA Gln is amidated to Gln-tRNA Gln. Table 15.1

8. Fig.15.7 tRNA elements that are recognized by aminoacyl- tRNA synthetases: the second genetic code The acceptor stem and the anticodon loop are the main parts of the tRNA molecule that are recognized by the aa- tRNA synthetase. Changing one nucleotide in the acceptor stem (the discriminator base) may be enough for the tRNA to be used by another synthetase.

9. Fig.15.8 Cocrystal structure between glytaminyl- tRNA synthetase and tRNA Gln The tRNA molecule is purple, the enzyme is gray and Glu-AMP is colored yellow, red and green. Note how close this molecule is to the 3’ end and the contact points between tRNA and synthetase.

10. Fig.15.9 Distinguishing features of similar amino acids The selection of an amino acid is a very precise process, less than 1 in 1000 tRNAs is charged with the incorrect amino acid. Some aa-tRNA synthetases proofread their product. Isoleucyl-tRNA synthetase has an editing pocket near its catalytic pocket. AMP-valine is hydrolyzed in this pocket while AMP-isoleucine is too large to fit.

11. Fig.15.10 The ribosome is unable to distinguish between correctly and incorrectly charged tRNAs: Cysteinyl-tRNA charged with cysteine or alanine is read by the ribosome as cysteine Cys

12. Fig.15.11 Prokaryotic RNA polymerase and ribosomes at work on the same mRNA Ribosome: 2-20 aa/sec RNA Pol: 50-100 nt/sec (DNA Pol: 200-1000 nt/sec). In eukaryotes, mRNA synthesis and protein synthesis take place in different cellular compartments. No need to keep up with RNA polymerase, so the ribosome incorporates 2-4 aa/sec

13. Fig.15.12 Separation of ribosomal subunits by ultracentrifugation In eukaryotes: 40, 60, 80 S is an abbreviation of Svedberg, named after the inventor of the ultracentrifuge, Theodor Svedberg

14. Fig.15.13 Composition of prokaryotic and eukaryotic chromosomes Although there are far more ribosomal proteins than rRNAs in each subunit, more than two-thirds of the mass of the prokaryotic ribosome is RNA. The ribosomal proteins are small (15 kD on average, while the 16 and 23S rRNAs are large (330 daltons per base, almost 1000 kD for 23S rRNA).

15. Fig.15.14 Translation: The ribosome cycle

16. Fig.15.15 A polyribosome or polysome Each ribosome contacts about 30 nucleotides of mRNA, but the large size of the ribosome only allows a density of 1 ribosome for every 80 nt of mRNA. Even a small ORF of 1000 bases can bind more than 10 ribosomes. If not for polysomes, only 10 % of the ribosomes in a cell would be active at a given time, due to the low concentrations of mRNA molecules.

17. Fig.15.16 The peptidyl transferase reaction This transfer of the growing peptide chain from the peptidyl- tRNA to the aminoacyl-tRNA is catalysed by the peptidyl transferase center in the large ribosomal subunit

18. Fig.15.17 The ribosome: Ribosomal RNAs play both a structural and catalytic role Most ribosomal proteins are on the periphery of the ribosome. The peptidyl transferase and the decoding centers are composed almost entirely of RNA (50S subunit at the top, with purple proteins and grey RNA, 30S at the bottom with dark blue protein and greenish RNA)

19. Fig.15.18 The ribosome has three tRNA-binding sites The A (aminoacyl) site is the binding site for the aminoacylated tRNA, the P (peptidyl) site is the binding site for the peptidyl-tRNA and the E (exit) site is the binding site for tRNA released after transfer of the polypeptide chain to the aminoacyl-tRNA. The tRNA binding sites are formed on the interface between the large and the small subunit, and can span the distance between the peptidyl transferase center in the large subunit and the decoding center in the small subunit.

20. Figure 15-21 The polypeptide exit tunnel in the large 50S subunit rRNA: white, ribosomal proteins: yellow, red and gold: rRNA in the peptidyl transferase center.

21. Fig.15.22 The events of translation initiation: an overview This overview is valid for both prokaryotes and eukaryotes, but the details are different!

22. Fig.15.23 In prokaryotes, 16S rRNA interacts with the RBS to position AUG in the P site (but not always as perfectly as in this case)

23. Fig.15.24 N-formyl methionine is the first amino acid to be incorporated into a polypeptide chain (but only in prokaryotes!) After synthesis of the polypeptide, the formyl group is removed by a deformylase. Often, the N-terminal methionine is also removed by an aminopeptidase, as well as one or two additional amino acids

24. Fig.15.25 Translation initiation in prokaryotes: Three initiation factors direct the assembly of an initiation complex that contain mRNA and the initiator tRNA IF1: Prevents binding of tRNA to the portion of the small subunit that will become the A site IF2: A GTPase that interacts with the small subunit, IF1 and fMet-tRNA fMet. Prevents other tRNAs from associating with the small subunit. Also acts as an initial docking site for the large subunit, that activates the GTPase activity IF3: Blocks the small subunit from reassociating with the large subunit. Normally, charged tRNAs enter the ribosome in the A site, but during initiation, the charged initiator tRNA enters the P site directly.

25. Figure 15-26 Eukaryotic initiation: After dissociation of the ribosome, four initiation factors, eIF1, eIF1A, eIF3, eIF5 bind to the small subunit, preventing binding of the large subunit and of tRNA to the A site, analogous to IF1 and 3 in prokaryotes. Initiator-tRNA is escorted to the small subunit by eIF2, a three-subunit GTP-binding protein (the ternary complex) and placed in the P site. In eukaryotes, ribosomes are recruited to mRNA by the 5’ cap. Before binding to the ribosome, the cap-binding protein eIF4E binds to the cap. Then eIF4G and eIF4A are recruited, followed by eIF4B. eIF4B activates the RNA helicase activity of eIF4A, which removes secondary structures in the mRNA before the mRNA is delivered to the 43S preinitiation complex to form the 48S preinitiation complex

26. Figure 15-27 In prokaryotes, the mRNA is held in a circle by interactions between initiation factors, primarily eIF4G, and polyA- binding protein

27. Figure 15-28 In eukaryotes, the start codon is found by scanning downstream of the 5’ end of the mRNA.The start codon is identified by base pairing with the initiator tRNA. eIF1 is released, eIF5 changes conformation, leading eIF2 to hydrolyze the bound GTP. With GDP bound, eIF2 no longer binds the initiator-tRNA and is released together with eIF5. This allows binding of eIF5B-GTP, promoting binding of the 60 S subunit.

28. Comparison between prokaryotic and eukaryotic initiation factors IF1 and eIF1 both bind to the A site to prevent interactions with tRNA. The function of IF2 is split between eIF2 and eIF5B. All three are regulated by GTP/GDP. IF3 and eIF1 both bind to the P-site, and both are released upon base pairing between initiator tRNA and AUG

29. Fig.15.29 The elongation steps of translation: a summary. Two helper proteins (elongation factors) are involved. The process is highly conserved between prokaryotes and eukaryotes, so only prokaryotic elongation is discussed here.

30. Fig.14.30 EF-Tu escorts aminoacyl-tRNA to the A site of the ribosome EF-Tu: GTP-binding protein with GTPase activity. EF-Tu-GTP binds to aminoacyl-tRNA, EF-Tu and EF-Tu- GDP has little affinity. The GTPase activity is stimulated by the same domain in the large subunit that stimulates the GTPase activity of IF-2 (the factor-binding center). Only after entrance of Aa-tRNA in the A site and formation of a correct codon-anticodon complex will the GTPase activity be stimulated

31. Figure 15-31 Three mechanisms ensure correct pairing between tRNA and mRNA: Additional pairing between two adjacent As in 16S rRNA in the A-site and the minor groove of correct base pairs formed between anticodon and the first two bases of the codon. To release EF-Tu, its GTP must be hydrolysed. Mismatches in the codon- anticodon pairing alter the position of EF-Tu, preventing its interaction with the factor-binding center and reducing its GTPase activity. After release of EF-Tu, the tRNA must rotate the aa towards the P-site in a process called accommodation. Incorrectly paired tRNA will often dissociate in this process.

32. Figure 15-33 Peptide bond formation: proposed role of the 2’OH group of tRNA Removal of the 2’-OH of the A residue at the 3’ end of the tRNA in the P-site reduces the reaction rate one million times. This figure shows a proposed “proton shuttle” mechanism to explain this.

33. Components of translation: mRNA (template) Amino acids (units to synthesize a polypeptide) Transfer RNAs Aminoacyl-tRNA synthetases Ribosomes (ribosomal RNAs + ribosomal proteins) Translation factors (initiation, elongation, termination)

34. Steps in translation Initiation (different in pro- and eukaryotes) Elongation (very similar in pro- and eukaryotes) Termination (very similar in pro- and eukaryotes)

35. Figure 15-34 The translocation reaction is stimulated by the elongation factor EF-G and requires GTP hydrolysis. After transfer of the peptide chain, the tRNA in the P-site prefers to bind in the E-site of the large subunit, while the now peptide- loaded tRNA in the A-site prefers the E-site. This is accompanied by a rotation of the small subunit. EF-G-GTP binds to this hybrid state and stabilizes it, but the contact between EF-G-GTP and the factor-binding center leads to hydrolysis of GTP. This changes the conformation of EF-G. “Gates” that separate the A-, P- and E-sites are opened, unlocking the ribosome, and EF-G- GDP is bound to the A-site. The A-site tRNA is moved fully to the P-site, pushing the P- site tRNA to the E-site and then out. The mRNA is moved 3 nucleotides due to the base pairing with the tRNA. The small subunit rotates back, EF-G-GDP no longer will bind to the A-site, and a new aa-tRNA can come in

36. Figure 15-35 EF-Tu-”GTP” and EF-G-GDP both bind to the A-site and have similar structures

37. Figure 15-36 In the process, EF-Tu-GTP and EF-G- GTP are hydrolysed to EF-Tu-GDP and EF-G-GDP. For the factors to participate in a new elongation cycle, GDP must be exchanged with GTP. For EF-G, the affinity for GTP is much higher than for GDP, so the nucleotide can easily be exchanged. EF-Tu needs the help of an exchange factor, EF-Ts

38. Fig.15.37 Release factors terminate translation in response to stop codons: the binding of RF1 to the A site GGQ motif Stop codons are recognized by class I release factors (RFs). In prokaryotes, RF1 recognizes UAG and RF2 UGA, while the third stop codon, UAA, is recognized by both. In eukaryotes, one single RF recognizes all three. Class II RFs (regulated by GTP binding and hydrolysis) stimulate the dissociation of the class I factors after release of the polypeptide chain

39. Fig.15.37 and 38 RF1 resembles a tRNA molecule Termination of translation When a stop codon UAG, UGA, UAA) enters the A-site, a release factor (RF) will recognize it and trigger a release of the peptide chain. There are two classes of RFs. Class I factors recognize the stop codon (in E. coli, UAG is recognized by RF1, UGA by RF2, and UAA by both). These factors bind in the A-site with their peptide anticodon (three amino acids) close to the stop codon.

40. Figure 15-39 When the peptide chain has been released, the class II release factor (RF3, eRF3) helps the dissociation of RF1/2. The class II proteins are GTP binding/hydrolysing proteins, like EF-G, IF2 and EF-Tu

41. Figure 15-40 Ribosome recycling factor (RRF) cooperates with EF-G and IF3 to recycle the ribosome after release of the peptide chain RRF binds in the A-site by mimicking a tRNA. EF-G- GTP is recruited by RRF and removes the tRNAs in a similar way to what happens in elongation. Then EF-G-GDP and RRF are released together with the mRNA.

42. Figure 15-41 Termination in eukaryotes The class I RF eRF1 acts like prokaryotic RF1 and RF2, but recognizes all three stop-codons. The class II RF eRF3-GTP delivers eRF1 to the ribosome. If eRF1 recognizes a stop codon, eRF3- GTP binds to the factor recognition center, leading to hydrolysis of GTP. eRF3-GDP is released and eRF1 moves into the peptidyl transferase center. No ribosome recycling factors in eukaryotes. Apparently, eRF1 together with the ATPase Rli1 take part in the preparation of the ribosome for a new translation event.

44. Regulation of translation Global regulation in eukaryotes: Phosphorylation of eIF2 Inactivation of eIF-4E Gene-specific regulation: Examples

45. Fig.15.45 Global regulation of initiation of eukaryotic translation by eIF4E- binding proteins Sometimes it is useful for eukaryotic cells to reduce translation globally. Two early stages are targeted for inhibition: recognition of mRNA, and initiator tRNA binding to the 40S subunit. 1) eIF2-GTP delivers initiator tRNA to the P-site. Phosphorylation of the α subunit of eIF2 inhibits a GTP- exhange factor for eIF2, called eIF2B, leading to reduced level of eIF2-GTP. 2) In translation initiation, eIF4E binds to the cap, and then to eIF4G. Other proteins, 4E-BPs (binding proteins), compete with eIF4G and act as inhibitors of translation initiation. If 4E-BPs are unphosphorylated, they bind tightly to eIF4E, while phosphorylation of BPs inhibit binding. The α subunit of eIF2 is phosphorylated by a number of kinases that are activated by conditions like amino acid starvation, viral infection and elevated temperature. mTor, that phosphorylates 4E-BPs, is activated by growth factors, hormones and other factors that stimulate cell division.

46. Regulation of translation Global regulation in eukaryotes: Phosphorylation of eIF2 Inactivation of eIF-4E Gene-specific regulation: Examples

47. Fig.15.46 Control of translation by mRNA-specific 4E-BPs: Cup specifically inhibits Oskar translation The Oskar protein is carefully located to the posterior regions of the Drosophila oocyte prior to fertilization. Oskar mRNA is not produced by the oocyte itself, but by attached nurse cells that deposit the mRNA in the anterior part of the oocyte. Then the mRNA is transported to the posterior part. During transport, translation of the mRNA is prevented by the action of a 4E-BP called Cup. Cup is recruited to the mRNA by another protein, Bruno, that binds to several sequences in the 3’-untranslated region. There is too little of Cup to have an effect on all translation, but this localization to a specific mRNA gives efficient inhibition of translation of that particular mRNA. Similar mechanisms regulate the expression of some other proteins as well.

48. Fig.15.42 Regulation of prokaryotic translation: Inhibition of the binding of the 30S subunit by masking the RBS

49. Fig.15.42 Regulation of prokaryotic translation of genes for ribosomal proteins: Protein from the red gene binds mRNA close to the translation initiation sequence of one of the most 5’-proximal genes

50. Fig.15.43 Regulation of ribosomal protein expression

51. Fig.15.44 Ribosomal protein S8 binds 16S rRNA and its own mRNA Similar sequences are shaded in dark green. The dashed lines box off that region in 16S rRNA protected by the S8 protein.

52. Fig.15.47 Regulation of ferritin translation by iron Careful regulation of the iron level in the human body is essential. The iron- binding protein ferritin is the major regulator of the level and acts by storing and releasing iron in a controlled manner. It is critical that the ferritin level responds quickly to changes in the level of free iron in the body. ferritin mRNA

53. Translational control of the abundance of the transcriptional activator Gcn4 in yeast Gcn4 activates transcription of genes involved in amino acid biosynthesis

54. tmRNAs rescue stalled ribosomes

55. Defective mRNAs are degraded in eukaryotes by translation-dependent mechanisms: Nonsense-mediated decay Nonstop-mediated decay No-go-mediated decay

56. Nonsense-mediated decay

57. Nonstop and no-go- mediated decay