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

2. Translation: one language to another
 more complex than replication & txn
Many steps and many proteins
Must be fast (20 amino acid/sec)
Must be accurate

3. speed vs. accuracy

4. Fidelity of translation:
correct recognition of codons on mRNA
An amino acid itself cannot recognize codon:
A transfer is required: tRNA
tRNA: adapter molecule that binds to a
specific codon and brings an amino
acid for incorporation into
polypeptide chain

5. alanyl-tRNA:
First nucleic acid
to be sequenced
 76 ribonucleotides

6. Anticodon on alanyl-tRNA
is complementary to one
of the codons for alanine

7. General structure of tRNA:
* Cloverleaf
* Half of residues
are base-paired
* Many common
structural features
 why?

8. General structure of tRNA: 73-93 ribonucleotides Enzyme-modified bases
* prevent base pairs
* create hydrophobicity
* allow protein interxn
* allow codon recognition

9. General structure of tRNA: Half of nucleotides form ds helices
5 places without ds:
*acceptor stem
*TψC loop
*extra arm
*DHU loop
*anticodon loop
Different but
structurally similar

11. General structure of tRNA:
5’ pG
Activated amino acid
attached to 3’ A-OH
Anticodon (near
center of seq.)

12. 3D structure of
yeast phenyl-
alanyl-tRNA

13. 3D structure of
yeast phenyl-
alanyl-tRNA
Important properties:
1. L-shaped
2. 4 helices  2 ds
3. H-bond interactions
for nonhelical regions
4. 3-terminus: flexible ss
5. Anticodon loop:
exposed at other end
 A good adaptor

15. Linkage of amino acid to tRNA is crucial:
Amino acid-tRNA establishes genetic code
Activate amino acid for later peptide bond
formation
(peptide bond formation: unfavorable)

16. Activated intermediates:
Amino acid esters
carboxyl  2’ or 3’OH
of A (tRNA)
Aminoacyl-tRNA or
charged tRNA

17. Activation reaction of an amino acid
Catalyzed by specific
aminoacyl-tRNA synthetases
(or activating enzymes)
Amino acid + ATP
amionacyl-AMP + PPi

18. Or amionacyl-AMP

19. The second step of the reaction
amionacyl-AMP + tRNA
aminoacyl-tRNA + AMP
Sum of reaction
Amino acid + ATP + tRNA + H2O
aminoacyl-tRNA + AMP + 2Pi

20. * Equivalent of 2 ATP is consumed in activation
* Activation and transfer steps for a particular
amino acid are catalyzed by the same amino-
acyl-tRNA synthetase
(intermediate does not dissociate from enzyme,
stably bound to active site)
* Acyl adenylate intermediate (also in fatty acid
activation

22. How does aminoacyl-tRNA synthetase incoporate
the correct amino acid?

23. Specific structure of the
amino acid binding site
 zinc ion + Asp
other synthetase have
different active site
structures

24. Proofreading by amino- acyl-tRNA synthetase
what happens when:
threonyl-tRNA synthetase
+ Ser-tRNAThr
 serine + tRNA
 editing: hydrolysis of
wrong amino acid
(carried out by editing
site; size exclusion)

25.  editing: hydrolysis of wrong amino acid
(carried out by editing site; size exclusion)
Most synthetases contain both editing site
& activation site
Activation (or acylation) site rejects larger
amino acids
Editing site cleaves activated amino acids that
are smaller than the correct one

26. Editing mechanism: The flexible CCA arm can
swing out of the activation
site and into the editing
site
Editing without
dissociating (fidelity )

27. Editing mechanism: High accuracy can still be achieved without editing
Proofreading: initial a.a.
binding interaction is not
good enough

28. How do synthetases choose their tRNA partners?
synthetases are the only molecules that “know”
the genetic code!
precise recognition of tRNAs
recognition is different for each synthetase-
tRNA

29. How do synthetases choose their tRNA partners?
Anticodon?
Some synthetases recognize their tRNA partners
primarily based on the anticodon loop.

30. Threonyl-tRNA synthetase
& tRNAThr
5’-CGU-3’  H bonds with G and U

31. How do synthetases choose their tRNA partners? microhelix in tRNA
A 24 nt microhelix can
be aminoacylated by
alanyl-tRNA synthetase
(without anticodon)
Mutated tRNACys
can be recognized by
synthetase (alanine)

32. Aminoacyl-tRNA synthetases:
diverse (independent evolution?)
Structural and sequence comparisons
They are related
Synthetases fall into two classes!

34. Differences between the two classes:
Different binding surfaces
CAA arm conformations
OH group acylation
ATP-binding conformations
Monomeric vs. dimeric

36. Ribosome: ribonucleoprotein particle with
large and small subunits
L1-L34
23S & 5S rRNA
S1-S21
16S rRNA

37. Ribosome: RNA is 2/3 of total mass 30S primary transcript 5S, 16S, 23S
Extensive folding
Internal base pairs
(conserved base-pairs,
not conserved seq.
ex. G-C vs. A-U)
16S rRNA 2o structure
* Defined structure
* Short duplex

38. “Chicken and egg” question:
If ribosomes synthesize proteins, where do
ribosomal protiens come from?
Protein: catalysis
RNA: structural OR
RNA: catalysis
Protein: structural and regulation

39. Proteins are synthesized in the N to C direction
How is mRNA read?

40. The direction of translation is 5’  3’

41. The direction of txn
is also 5’ to 3’
Same direction
So there is a coupling
between txn and tsl
Efficiency
Polyribosome or
polysome 

42. Translation initiation
The first codon is more than 25nt from 5’
Ribonuclease digestion: initiator region on mRNA 2 1

43. 3 ~ 9 bp
Two kinds of interactions determine tsl initiation

44. Translation is initiated by
formylmethionyl-tRNAf
tRNAf vs. tRNAm

45. Ribosomes have 3 tRNA-binding sites
Exit
Peptidyl
Aminoacyl

48. Tunnel for protein escape

49. Mechanism of protein synthesis
30S + mRNA
tRNAf  Met
50S

51. Peptidyl transferase center on 23S rRNA

52. Center: promote reaction & stabilize intermediate

55. The peptide chain remains in the P site on the 50S (tunnel)

56. The amino acid in the aminoacyl-tRNA does not
play a role in selecting a codon

58. Codon-anticodon interaction:
* Watson-crick base pairing
* Anti-parallel
* One anticodon for one codon?
Some tRNA recognize more
than one codon:
Alanyl-tRNA: GCU, GCC, GCA
Degeneracy of genetic code:
XYU & XYC
XYA & XYG

60. Wobble: steric freedom

61. Appears in several anticodon

62. Anticodon of yeast alanyl-tRNA: IGC
Codons: GCU, GCC, GCA

63. Codon-anticodon interactions:
The first two bases of codon:
standard pairing
 codons differ in the first 2 bases are
recognized by different tRNAs
(ex. UUA and CUA of leucine)
First base of anticodon determines how
many codons to be read
 degeneracy of genetic
code: from wobble interxn

64. Why wobble only in the third base of codon?
30S/16S rRNA:
A1492, A1493, G530
forms H bond with first
2 paired anticodon-codon
(check WC bp)
Ribosome plays an active
role in decoding codon-
anticodon interactions