1. Translation

2. 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. Structure of messenger RNAs of prokaryotes and eukaryotes
Kozak sequence

4. 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)

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

6. From the cloverleaf to the actual 3D structure

7. Coupling an amino acid to a tRNA molecule
Two steps:
Formation of an aminoacyladenylylate, a mixed acid anhydride that remains closely associated with the enzyme
Transfer of the aminoacyl residue to tRNA

9. 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.
Fig.15.7

10. Co-crystal structure between glytaminyl-tRNA synthetase and tRNAGln

11. 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.

12. Cys
The ribosome is unable to distinguish between correctly and incorrectly charged tRNAs: cysteinyl-tRNA charged with cysteine or alanine binds to a cysteine codon.
Cys

13. Prokaryotic RNA polymerase and ribosomes at work on the same mRNA
Ribosome: 2-20 aa/sec
RNA Pol: nt/sec
(DNA Pol: 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

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

15. Prokaryotic and eukaryotic ribosomes

16. The ribosome cycle

17. A polyribosome or polysome

18. 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

19. The ribosome: Ribosomal RNAs play both a structural and catalytic role

20. 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.

21. The polypeptide exit tunnel in the large 50S subunit
Figure 15-21
Figure 15-21
©2002 Macmillan
The polypeptide exit tunnel in the large 50S subunit
rRNA: white, ribosomal proteins: yellow, red and gold: rRNA in the peptidyl transferase center. 21

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

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

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

25. N-formyl methionine is the first amino acid to be incorporated into a polypeptide chain 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

26. Translation initiation in prokaryotes: Three initiation factors direct the assembly of an initiation complex that contains 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-tRNAfMet. 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.

28. 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
Figure 15-26 28

29. Assembly of the 43S preinitiation complex

30. Assembly of eIF4 at the 5’ cap of eukaryotic mRNAs

31. In eukaryotes the mRNA is held in a circle by interactions between initiation factors, primarily eIF4G, and polyA-binding protein.
Figure 15-27 31

32. 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.
Figure 15-28 32

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

34. The elongation steps of translation: a summary.
Two elongation factors are involved.
The process is highly conserved between prokaryotes and eukaryotes.

35. 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

36. 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.
Figure 15-31 36

40. Peptide bond formation is catalyzed by ribosomal RNA
Active site
Grey: ribosomal RNA
Purple: proteins

41. Peptide bond formation: proposed role of the 2’OH group of tRNA
Figure 15-33
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. 41

42. 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 P-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.
Figure 15-34 42

44. EF-G-GTP

45. EF-Tu-”GTP” and EF-G-GDP both bind to the A-site and have similar structures
Figure 15-35 45

46. 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.
Figure 15-36 46

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

48. Termination of translation
recycling
Table Genomes 3 (© Garland Science 2007)

49. Release factors terminate translation at a stop codon
Peptide anticodon
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.
GGQ motif

50. 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.
Figure 15-39 50

51. 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 as in elongation. EF-G-GDP and RRF dissociate from the ribosome allowing binding of IF3 which prevents association of small and large ribosomal subunits. Finally, RRF and the mRNA are released.
Figure 15-40 51

52. 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 contacts the factor-binding center, leading to hydrolysis of GTP. eRF3-GDP is released and the GGQ motif on eRF1 moves into the peptidyl transferase center where it cleaves the polypeptide from the tRNA.
No ribosome recycling factors in eukaryotes. Apparently, eRF1 together with the ATPase Rli1 take part in ribosome disassembly.
Figure 15-41 52

53. Figure 15-41 53

54. Regulation of translation
Global regulation in eukaryotes:
Phosphorylation of eIF2
Inactivation of eIF-4E
Gene-specific regulation:
Examples
Figure 15-41 54

55. Assembly of the 43S preinitiation complex
eIF2-GTP
The α subunit of eIF2 can be phosphorylated by a number of kinases that are activated by conditions like amino acid starvation, viral infection and elevated temperature.

56. Global regulation of initiation of eukaryotic translation by eIF4E-binding proteins
4E-BPs compete with eIF4G for binding to eIF-4E. When bound to eIF-4E, 4E-BPs inhibit assembly of the cap-binding complex, resulting in inhibition of translation.
mTor kinase enhances translation by phosphorylating 4E-BPs, thereby preventing binding of 4E-BPs to eIF-4E. mTor kinase is activated by growth factors, hormones and other factors that stimulate cell division.

57. Regulation of translation
Global regulation in eukaryotes:
Phosphorylation of eIF2
Inactivation of eIF-4E
Figure 15-41
Gene-specific regulation:
Examples 57

58. 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.

59. Regulation of prokaryotic translation: Inhibition of the binding of the 30S subunit by masking the RBS

60. 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

61. Regulation of ribosomal protein expression

62. Ribosomal protein S8 binds 16S rRNA and its own mRNA
Identical sequences are shaded in dark green. The dashed lines box off the region in 16S rRNA protected by the S8 protein.

63. Regulation of ferritin translation by iron
ferritin mRNA
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.

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

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

66. tmRNAs rescue stalled ribosomes

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

68. Nonsense-mediated decay

69. Nonstop and no-go- mediated decay

70. Subunits of DNA polymerase III in E. coli.
Figure 13.21a Genomes 3 (© Garland Science 2007)

71. Figure 13.21b Genomes 3 (© Garland Science 2007)

72. Figure 13.21c Genomes 3 (© Garland Science 2007)

73. Processing of polypeptides
Figure Genomes 3 (© Garland Science 2007)

74. Figure 13.25 Genomes 3 (© Garland Science 2007)

75. Figure 13.26 Genomes 3 (© Garland Science 2007)

76. In addition: Hsp40 (stimulates ATPase activity) GrpE (removes ADP)
Figure Genomes 3 (© Garland Science 2007)

77. GroEL/GroES complex (chaperonins in bacteria and eukaryotes)
GroES (7 subunits)
GroEL
(14 sub-
units)
Figure Genomes 3 (© Garland Science 2007)

78. Figure 13.24 Genomes 3 (© Garland Science 2007)

79. Figure 13.29 Genomes 3 (© Garland Science 2007)

80. Processing of promelittin (venom of bees)
of an extracellular protease
Figure Genomes 3 (© Garland Science 2007)

81. Processing of insulin 105 aa
Figure Genomes 3 (© Garland Science 2007)

82. Peptide hormones in vertebrates
Produced in pituitary gland
Processing different in different cell types
Figure Genomes 3 (© Garland Science 2007)

83. Figure 13.24 Genomes 3 (© Garland Science 2007)

84. Table 13.6 Genomes 3 (© Garland Science 2007)

85. Histone H3 modifications
Figure Genomes 3 (© Garland Science 2007)

86. Serine or threonine
Figure 13.34a Genomes 3 (© Garland Science 2007)

87. Asparagine
Figure 13.34b Genomes 3 (© Garland Science 2007)

88. Figure 13.24 Genomes 3 (© Garland Science 2007)

89. First amino acid of extein is cysteine, serine, or threonine
(autocatalytic)
Figure Genomes 3 (© Garland Science 2007)

90. Intein homing Sequence-specific endonuclease
Figure Genomes 3 (© Garland Science 2007)

91. Eukaryotic proteasome
14 different subunits
Ubiquitinated proteins enter
the cylinder unfolded and are degraded to short peptides.
(26S)
(19S)
(Ubiquitin is a small protein of 76 aa.)
Figure Genomes 3 (© Garland Science 2007)