1. Biochemistry Sixth Edition
Berg • Tymoczko • Stryer
Chapter 30:
Protein Synthesis
Copyright © 2007 by W. H. Freeman and Company

2. Protein Chain Growth
Aminoacyl tRNA is the carrier.

4. DNA Code
Each amino acid corresponds to a three letter codon present in the coding strand of dsDNA. Letters are GCAT and there are 43 = 64 possible three base sequences. There are 61 for the amino acids and 3 stop codons called nonsense sequences. Procaryotes and eucaryotes use the same code.
There are no spaces between the three base codons (unpunctuated) and no signals between codons (non-overlapping). The code is degenerate but unambiguous, i.e. more than one codon for the same amino acid but 1 codon = 1 AA.

5. DNA Code
Codons exhibit “wobble” (flexibility in the third base, the 5' end of the anticodon pairs with the 3' end of the codon).
Wobble relationships: ' anticodon 3' codon
Anticodon 5'…C-A-U…3' I U, C, A Codon 3'…G-U-A…5' G U, C U A, G Table A U C G

6. DNA Code
There is only one code for Met: AUG which is the initiation codon for protein synthesis.
Termination sequences:
UAA = ochre; UAG = amber; UGA = opal
Amber was the first to be discovered, by Harris Bernstein of Cal Tech. It was called amber after Bernstein which means amber in German. Others were named to follow the color metaphor.

7. Transfer RNA
tRNA has a cloverleaf structure with ~73-93 residues (MW = D). Pretranscripts have ~ residues. Eucaryotes transport tRNA to the cytosol for processing. Base modification of ~15% is common but can be as high as 40%.
Numbering begins at the 5' end. ~85% have G at the 5' end and all have CCA at the 3' end.
Each tRNA has a “D” loop, an anti-codon loop and a TψC loop, in some there is a small variable loop between the anticodon loop and the TψC loop. Duplex RNA maintains the loops.

8. Yeast Ala tRNA Shows a number of modified bases.
UH2 Dihydrouridine I Inosine ψ pseudouridine mI methyl inosine mG methyl guanine m2G dimethyl guanine T ribothymine

9. tRNA-mRNA
The numbering here should be reversed to show the normal codon: anticodon view.
Base pairing for yeast Ala tRNA.

10. Base Modification
Methylation Reduction.

11. General tRNA Structure
Shows the significant parts of tRNA.

12. Space filling Model Colors match the previous planar representation.
Shows L-shape.

13. Skeletal Model
Phe tRNA.
Shows L- shape.

14. Aminoacyl tRNAs
In order for peptide bond formation to be favorable thermodynamically, the carboxyl group of the amino acid must be activated.
Activation of amino acids is catalyzed by aminoacyl tRNA synthetases. There are about 20 of these (one for each amino acid) that catalyze joining the carboxyl group of a specific amino acid to the 3' OH of a specific tRNA through an ester bond.
This is a two step process. Aminoacyl-AMP is first formed as an intermediate followed by formation of aminoacyl-tRNA.

15. Aminoacyl tRNAs 1. Formation of aminoacyl-AMP:
AA + ATP -- > AA-AMP + PPi
The aminoacyl-AMP is a mixed anhydride and the PPi goes to 2 Pi.
2. Formation of aminoacyl-tRNA:
AA-AMP + tRNA -- > AA-tRNA + AMP
The mixed anhydride reacts with the 3'OH (an alcohol) to give and ester and and acid (AMP).
Net: AA + ATP + tRNA -- > AA-tRNA + AMP + 2 Pi

16. Activated Amino Acid
An amino acid is attached to 3' end of tRNA as an aminoacyl group.
Some attachments are made to the 3' OH and others are to the 2' OH. These later move to the 3' OH.

18. Similar Structures
Thr, Ser and Val have side chains that are spatially similar.
How are specific match-ups of the correct AA and tRNA made?

19. Thr tRNA Synthetase
Zn++ binding to α-NH2 and the side chain OH excludes Val binding.
Mistakes occur with Ser.

20. Error Correction
Ser bound to Thr tRNA is recognized by the editing site in the synthetase and Ser is hydrolyzed.
This occurs without dissociation from the enzyme.

21. Model of tRNA and Synthetase
Shows proximity of the active site to the editing site.

22. Structural Model
The Thr tRNA -synthetase complex.

23. Abbreviated Structure
Directs the correct amino acylation without an anti-codon loop.
Indicates that not all synthetases require the anti-codon.

24. Classes of Synthetases
Synthetases fall into two classes, each is specific for 10 of the amino acids.
Class I enzymes tend to be monomers, the CCA is in a hairpin structure and they acylate the 3' OH.
Class II enzymes tend to be dimers, the CCA is in a helical structure and they acylate the 2' OH.
Also, enzymes of each class bind to a different face of tRNA.

27. Ribosomes
These are ribonucleic acid-protein assemblies that associate tRNA, mRNA and synthesize protein. The generally have an rRNA core coated with proteins.
Procaryotes have ~104 ribosomes/cell whereas eucaryotes have ~107 ribosomes/cell.
About 50% of the dry weight of bacterial cells is ribosome.
Each ribosome has a peptidyl site (P site) and an aminoacyl site (A site).

28. General Ribosome Structure
Procaryotic Eucaryotic
70S S S 30S 60S 40S 23S RNA 16S RNA 28S RNA 18S RNA 5S RNA S RNA S ~31 ~21 ~40 ~ proteins proteins

29. Procaryotic Ribosome

30. 16S rRNA
Secondary and tertiary structure.

31. Direction of Translation
Synthetic messenger
Protein product

32. E.coli
Transcription and translation occurring.

33. Initiation This is the rate limiting step of translation.
The initiation start site is determined both by specific binding of mRNA to the 3' end of the 16S rRNA and by the codon/anticodon base pairing relationship beetween mRNA and the initiator tRNA.
The initiator codon in E.coli is AUG and is at the 5' end of mRNA just downstream from a conserved purine rich sequence, GGAG, called the Shine-Delgarno sequence. So, the first AUG may not be the initiator codon.

34. Initiation Sequences
Bacterial and viral.

35. E.coli Initiation
The initiator, Met-tRNAf , is used only at the initiator codon. A different tRNAm is used for internal Met but both recognize AUG. After formation of Met-tRNAf by synthetase it is formylated by a formyl transferase using N10-formylFH4.
Met-tRNAf + N10-formylFH4. -- > formylMet-tRNAf + FH4
When translation gets underway, the N-term formylMet is deformylated or removed using a deformylase enzyme or an aminopeptidase, resspectively.

36. FormylMet-tRNAf
Other Met uses tRNAm.

37. Ribosome Binding Sites
E for exit, P for peptidyl and A for aminoacyl.

38. Another View
mRNA is within the 30S subunit and the E, P and A sites span both 30S and 50S subunits.
Only tRNA in the P & A sites bind to mRNA.

39. Initiation Events
There are a number of proteins that serve as participate in initiation, elongation and termination. In E.coli there are three pprotein initiation factors, IF1, IF2, and IF3. These operate only during initiation events. Although they bind on the ribosome surface during initiation they are not ribosomal proteins.
An initiation complex is formed between the 30S rRNA, the IFs, fMet-tRNAf and mRNA.

40. Initiation Events
30S + IF1 + IF3 -- > 30S•IF1•IF3 (IF3 prevents 50S binding until fMet-tRNAf and mRNA are bound)
IF2 with GTP seeks and binds fMet-tRNAf then combines with 30S•IF1•IF3.
IF2 + GTP -- > IF2•GTP + fMet-tRNAf -- > IF2•GTP•fMet-tRNAf
IF2•GTP•fMet-tRNAf + 30S•IF1•IF3 -- > IF2•GTP•fMet-tRNAf•30S•IF1•IF3

41. Initiation Events
mRNA now comes in and binds correctly causing a conformational change that expels IF1 and IF3.
IF2•GTP•fMet-tRNAf•30S•IF1•IF3 + mRNA -- > IF2•GTP•fMet-tRNAf•30S•mRNA + IF1 + IF3
IF2 promotes association of the 50S subunit, GTP hydrolyzes to GDP and IF2 dissociates. This yields the 70S initiation complex.
IF2•GTP•fMet-tRNAf•30S•mRNA + 50S -- > IF2•GDP + fMet-tRNAf •30S•mRNA•50S

43. Elongation Events
After the initiation events fMet-tRNAf is in the P-site and the other two sites are empty.
A peptide is synthesized from N-term to C-term in a three step process (~18 residues/sec).
1. A GTP dependent, codon guided binding of aminoacyl tRNA to the A-site Transfer of the peptidyl strand in the P-site to the new AA tRNA in the A-site Movement of the empty tRNA from P to the E-site and dissociation. Translocation of the peptidyl tRNA from A to P and movement of 70S along mRNA to the next codon.

44. Elongation Events
E.coli uses only 3' tRNA esters in elongation. It also uses three elongation factors, EF-TU, EF-TS and EFG (a translocase).
Step 1. EF-TU needs GTP, is responsible for getting the correct AA tRNA into the A-site and will bind to any AA tRNA except initiator.
EF-Tu + GTP -- > EF-Tu•GTP
EF-Tu•GTP + AA tRNA -- > EF-Tu•GTP•AA tRNA
EF-Tu•GTP•AA tRNA + A-site -- >
EF-Tu•GTP•AA tRNA•A-site (if matched)

45. EF-Tu binding tRNA

46. Elongation Events
If a match is made then GTP hydrolyzes and EF-Tu•GDP dissociates. GTP will not hydrolyze unless a codon/anticodon match is made. Now, a new GTP needs to exchange for GDP to reactivate EF-Tu, this requires EF-Ts.
EF-Tu•GDP + EF-Ts -- > EF-Tu•EF-Ts + GDP
EF-Tu•EF-Ts + GTP -- > EF-Tu•GTP + EF-Ts
Step 2. Peptidyl transferase activity catalyzes movement of the peptidyl chain to the amino group of the AA tRNA in the A-site. This enzyme is a ribozyme and does not use ATP.

47. Elongation Events
Step 3. Movement tRNA from site P to E and A to P as well as translocation along mRNA (three residues) requires elongation factor G (EFG) and GTP. GTP hydrolysis promotes movement along mRNA and concommitant change in site position. As GTP hydrolyzes EFG•GDP dissociates from 70S.
EFG + GTP -- > EFG•GTP
EFG•GTP + 70S (P & A occupied) -- > complex > EFG•GDP + 70S (E & P occupied)
Another GTP displaces GDP from EFG.

48. Elongation Model

49. Elongation Reaction

50. Movement on mRNA
Movement on mRNA & Binding EFG•GTP Hydrolysis of GTP

51. Termination Events
E.coli uses three release factors, RF1, RF2 and RF3. The termination or stop codon (UAG, UGA or UAA) is on the 3' end of mRNA and these usually occur in pairs. When one of these codons appears in the A-site, translation ceases and one of the RFs enters the A-site.
RF1 if UAG or UAA. RF2 if UGA or UAA. RF3 binds GTP and augments RF1 and RF2.
The peptide is hydrolyzed from the P-site along with hydrolysis of GTP.
If not done previously, the N-term is deformylated or removed.

52. Removal of the Peptide.
Hydrolysis from the P-site.

53. Termination Events
Binding RF Cleavage of at A-site Peptide

54. Energy Cost per residue Activation ATP -- > AMP 2 Synthetase
Elongation GTP -- > GDP 1 EF-Tu, step GTP -- > GDP 1 EFG, step 3
per protein
Initiation GTP -- > GDP 1 IF2, 70S
Termination GTP -- > GDP 1 RF3

55. Processing the Protein
The 70S ribosomal tunnel is ~30 residues long.
As the protein exits the ribosome, modification may begin. Cotranslational modifications occur before translation is complete and those after completion are posttranslational. Examples:
Hydroxylation 4HPro, 5HLys Methylation MeLys, MeGlu Amidination Asn, Gln, C-term Phosphorylation Ser, Thr, Tyr Disulfide formation Cys Iodination Tyr

56. Eucaryotic Translation
Ribosomes are larger (80S).
There are more initiation factors (eIFs). eIFs and polyA binding protein promote formation of a circular mRNA.
Met is not formylated. The first AUG codon is normally the start codon and there is no Shine-Delgarno sequence. Some use a start signal.
There are three elongation factors similar to those in E.coli.
There is a single release factor (RF) which recognizes all three stop codons.

58. Biochemistry Sixth Edition
Berg • Tymoczko • Stryer
End of Chapter 30
Copyright © 2007 by W. H. Freeman and Company