1. Molecular Biology Fourth Edition
Chapter 17
The Mechanism of Translation I: Initiation
Chapter 18
The Mechanism of Translation II: Elongation and Termination
Chapter 19
Ribosomes and Transfer RNA
Molecular Biology Fourth Edition
Robert F. Weaver

2. 17.1 Initiation of Translation in Bacteria
Two important events must occur before translation initiation can take place
Generate a supply of aminoacyl-tRNAs
Amino acids must be covalently bound to tRNAs
Process of bonding tRNA to amino acid is called tRNA charging
Dissociation of ribosomes into their two subunits
The cell assembles the initiation complex on the small ribosomal subunit
The two subunits must separate to make assembly possible

3. tRNA Charging All tRNAs have same 3 bases at 3’-end (CCA)
Terminal adenosine is the target for charging with amino acid
Amino acid attached by ester bond between
Its carboxyl group
2’- or 3’-hydroxyl group of terminal adenosine of tRNA
Amino acid

4. Two-Step Charging
Aminoacyl-tRNA synthetases join amino acids to their cognate tRNAs
This is done in a two-step reaction:
Begins with activation of the amino acid with AMP derived from ATP
In the second step, the energy from the aminoacyl-AMP is used to transfer the amino acid to the tRNA

5. Aminoacyl-tRNA Synthetase Activity
AMP/amino acid coupling
AMP/tRNA displacement

6. Dissociation of Ribosomes
E. coli ribosomes dissociate into subunits at the end of each round of translation
IF1 actively promotes this dissociation
IF3 binds to free 30S subunit and prevents reassociation with 50S subunit to form a whole ribosome

7. Ribosomal Subunit Exchange
Grow in heavy isotope of nitrogen, carbon, and hydrogen. Then 3H labeled

8. Formation of the 30S Initiation Complex
When ribosomes have been dissociated into 50S and 30S subunits, cell builds a complex on the 30S subunit:
mRNA
Aminoacyl-tRNA
Initiation factors
IF3 binds by itself to 30S subunit
IF1 and IF2 stabilize this binding
IF2 can bind alone, but is stabilized with help of IF1 and IF3
IF1 does not bind alone

9. First Codon and the First Aminoacyl-tRNA
Prokaryotic initiation codon is:
Usually AUG
Can be GUG
Rarely UUG
Initiating aminoacyl-tRNA is N-formyl-methionyl-tRNA
N-formyl-methionine (fMet) is the first amino acid incorporated into a polypeptide
This amino acid is frequently removed from the protein during maturation

10. N-Formyl-Methionine
Lipman et al.,
Marcker and Sanger

11. Formyl-Met-tRNA and Met-tRNA
Which codons they respond to?
Which position within the protein they placed methionine?

12. Which codons they respond to?
Make a labeled aminoacyl-tRNA, mix it with ribosomes and a variety of trinucleotides, such as AUG.
Met-tRNA bind AUG, formyl-Met-tRNA binds AUG, GUG, and UUG.
AUG >90% , GUG about 8% UUG 1%

13. Which position within the protein they placed methionine?
mRNA Sequence AUG AUG AUG…….
In vitro translation system
In the presence of tRNAmet, met is incorporated into interior of the product
In the presence of formy-tRNAmet, met is incorporated into the first codon of the product

14. Weigert and Garen: Formyl-Met of the the polypeptide is always removed in bacteria and phage proteins

15. Binding mRNA to the 30S Ribosomal Subunit
The 30S initiation complex is formed from a free 30S ribosomal subunit plus mRNA and fMet-tRNA
Binding between the 30S prokaryotic ribosomal subunit and the initiation site of a message depends on base pairing between
Short RNA sequence
Shine-Dalgarno sequence
Upstream of initiation codon
Complementary sequence
3’-end of 16S RNA

16. Binding mRNA and fMet-tRNA to the 30S ribosomal subunit
How does the cell detect the difference between the initiation codon and an ordinary codon with the same sequences?
Consensus sequences?

17. Positive strand phagesf2
MS2
Positive strand phages
Encode three genes: A (maturation) protein, coat protein and replicase

18. Gene structure of MS2 phage RNA

19. The secondary structures have inhibitory effect

20. Lodish et al:
Translating f2 coat mRNA by ribosomes from different bacteria
B. stearothermophilus could only translate A protein but not coat protein
(This is not due to initiation factors but ribosomes)

21. The important element is in the 30S ribosome
Nomura et al.,
The important element is in the 30S ribosome
If the 30S ribosome from the E. coli, the coat protein can be translated
If the 30S ribosome from the B. stearothermophilus, the coat protein can not be translated
The active elements are S12 and 16S rRNA

22. Shine and Dalgarno
Upstream of initiation codon: 5’- AGGAGGU
3’ end of the 16S rRNA: 3’HO-AUUCCUCCAC
B. stearothermophilus has poor match

23. Bacillus 16S rRNA
4 Watson Crick base pairing with the A protein and replicase ribosome binding sites
2 with the coat protein gene
E. coli
At least three base pairs with all three genes
Could the base pairing between 16S rRNA and
the region upstream of the translation initiation
site be vital to ribosome binding?

24. Shine-Dalgarno (SD) sequence
See Table 17.1
AGGAGGU
From ribosomes from C. crescentus and P. aeruginosa
No ribosome binding would occur for less than 3 bp

26. Steitz and Jakes
Ribosome from E. coli bound to initiation site and treated with Colicin E3 (RNase)
Fingerprinting
Initiation site including S-D sequence
An oligonucleotide from 3’end of the 16S rRNA

27. The best evidence Translation Hui and De Boer in 1987
Clone the human growth hormone gene into E. coli
Translation
WT SD sequence
WT 16S rRNA
GGAGG
CCUCC (5 pairing) OK
SD sequence
16S rRNA
CCUCC
CCUCC (no pairing) NO
GUGUG
CCUCC (2 pairing)
GGAGG (5 pairing)

28. Initiation Factors and 30S Subunit
Binding of the Shine-Dalgarno sequence with the complementary sequence of the 16S rRNA is mediated by IF3
Assisted by IF1 and IF2
All 3 initiation factors have bound to the 30S subunit at this time

29. Binding of fMet-tRNA to the 30S Initiation Complex
IF2 is the major factor promoting binding of fMet-tRNA to the 30S initiation complex
Two other initiation factors also play an important supporting role
GTP is also required for IF2 binding at physiological IF2 concentrations
The GTP is not hydrolyzed in the process

30. 30S Initiation Complex
The complete 30S initiation complex contains one each:
30S ribosomal subunit
mRNA
fMet-tRNA
GTP
Factors IF1, IF2, IF3

31. Formation of the 70S Initiation Complex
GTP is hydrolyzed after the 50S subunit joins the 30S complex to form the 70S initiation complex
This GTP hydrolysis is carried out by IF2 in conjunction with the 50S ribosomal subunit
Hydrolysis purpose is to release IF2 and GTP from the complex so polypeptide chain elongation can begin

32. What is the function of GTP hydrolysis
GTP hydrolysis is to remove IF-2 from the ribosomes
Exp: 30S initiation complex+labeled IF-2 and fMet-tRNA and either GDPCP or GTP, add 50S and ultracentrifugation

33. 17.18 Effect of GTP hydrolysis on release of IF-2 from the ribosome.
After adding 50S, IF-2 is released from the 70S ribosome but fmet-tRNA is still associated

34. In Fig 17.18, more fMet-tRNA is associated with the 70S ribosomes
Catalytic function of IF2
Hydrolysis of GTP is necessary to release IF2 from the 70S initiation complex so it can bind another molecule of fmet-tRNA, otherwise, IF2 only acts stoichiometrically

35. Bacterial Translation Initiation
IF1 influences dissociation of 70S ribosome to 50S and 30S
Binding IF3 to 30S, prevents subunit reassociation
IF1, IF2, GTP bind alongside IF3
Binding mRNA to fMet-tRNA forming 30S initiation complex
Can bind in either order
IF2 sponsors fMet-tRNA
IF3 sponsors mRNA
Binding of 50S with loss of IF1 and IF3
IF2 dissociation and GTP hydrolysis

36. 17.2 Initiation in Eukaryotes
Eukaryotic
Begins with methionine
Initiating tRNA not same as tRNA for interior
No Shine-Dalgarno
mRNA have caps at 5’end
Bacterial
N-formyl-methionine
Shine-Dalgarno sequence to show ribosomes where to start

37. Scanning Model of Initiation
Eukaryotic 40S ribosomal subunits locate start codon by binding to 5’-cap and scanning downstream to find the 1st AUG in a favorable context
Kozak’s Rules are a set of requirements
Best context uses A of ACCAUGG as +1:
Purine (A or G) in -3 position
G in +4 position
5-10% cases ribosomal subunits bypass 1st AUG scanning for more favorable one

38. The scanning model of initiation
Kozak systematically mutated nucleotides around the initiation ↓
codon in a cloned rat preproinsulin gene
Introduce into COS cells
Label newly synthesized protein with 35S-Met
Immunoprecipitate
Electrophoresis
Detect by autoradipgraph

39. The scanning model of initiation
The better the translation initiation,
the more proinsulin was made

40. The best initiation occur with a G or an A in position –3 and a G in position +4 (where the A in ATG is position +1)
A/G C C A T G G

41. Figure 17.21 Effects of single base changes in positions –3 and +4 surrounding the initiating ATG.

42. If this really is the optimum sequence for translation initiation, introducing it out of frame and upstream of the normal initiation codon should provide a barrier to scanning ribosomes and force them to initiate out of frame

43. The closer it resembled the optimal sequence, the more it
Out-of-frame
ATG
The closer it resembled the optimal sequence, the more it
interfered with initiation at the downstream ATG

44. Translation With a Short ORF
Ribosomes can use a short upstream open reading frame:
Initiate at an upstream AUG
Translate a short Open Reading Frame
Continue scanning
Reinitiate at a downstream AUG

45. Scanning Model for Translation Initiation

46. Effects of mRNA Secondary Structure
Secondary structure near the 5’-end of an mRNA can have either positive or negative effects
Hairpin just past an AUG can force a pause by ribosomal subunit and stimulate translation
Very stable stem loop between cap and initiation site can block scanning and inhibit translation

47. Eukaryotic Initiation Factors
Bacterial translation initiation requires initiation factors as does eukaryotic initiation of translation
Eukaryotic system is more complex than bacterial
Scanning process
Factors to recognize the 5’-end cap

48. Translation Initiation in Eukaryotes
Eukaryotic initiation factors and general functions:
eIF2 binds Met-tRNA to ribosomes
eIF2B activates eIF2 replacing its GDP with GTP
eIF1 and eIF1A aid in scanning to initiation codon
eIF3 binds to 40S ribosomal subunit, inhibits reassociation with 60S subunit
eIF4 is a cap-binding protein allowing 40S subunit to bind 5’-end of mRNA
eIF5 encourages association between 60S ribosome subunit and 48S complex
eIF6 binds to 60S subunit, blocks reassociation with 40S subunit
Fig

49. Function of eIF4 eIF4 is a cap-binding protein
This protein is composed of 3 parts:
eIF4E, 24-kD, has actual cap binding activity
eIF4A, a 50-kD polypeptide
eIF4G is a 220-kD polypeptide
The complex of the three polypeptides together is called eIF4F

50. The cap-binding proteins
GDP-binding protein
Cap binding protein

51. Function of eIF4A and eIF4B
Has an RNA helicase activity
This activity unwinds hairpins found in the 5’-leaders of eukaryotic mRNA
Unwinding activity is ATP dependent
eIF4B
Has an RNA-binding domain
Can stimulate the binding of eIF4A to mRNA

52. Function of eIF4G
eIF4G is an adaptor protein capable of binding to other proteins including:
eIF4E, cap-binding protein
eIF3, 40S ribosomal subunit-binding protein
Pab1p, a poly[A]-binding protein
Interacting with these proteins lets eIF4G recruit 40S ribosomal subunits to mRNA and stimulate translation

53. Inhibit capped cellular mRNA
Fig
Inhibit capped cellular mRNA
Synergy effect
Regulatory protein and miRNA bound to the 3’UTR are close to the cap, which could help them influence the cap
2. Ribosomes copleteing one round of translation are close to the cap
3. Unavailable to RNase

54. Structure and Function of eIF3
eIF3 is a 5-lobed protein that binds at the same site to:
eIF4G
Prominent part of viral IRES
This explains how the IRES can substitute for 40S ribosomal subunit to mRNA
Cryo-EM studies have produced a model for the eIF3-IRES-40S complex explaining how eIF3 prevents premature 40S-60S association (sits at the site of proposed key contact between two particles)

55. Prevention of Premature 40S-60S Association
eIF3 blocks key contact point between subunits 40S and 60S
eIF4G, so also eIF4E, locate close to the cap on an mRNA bound to 40S ribosomal particle
eIF4 would be in position to cap-bind

56. Functions of eIF1 and eIF1A
eIF1 and eIF1A act synergistically to promote formation of a stable 48S complex involving:
Initiation factors
Met-tRNA
40S ribosomal subunits bound at initiation codon of mRNA
eIF1 and eIF1A act by
Dissociating improper complexes between 40S subunits and mRNA
Encouraging formation of stable 48S complexes

57. Principle of the Toeprint Assay
Source: Adapted from Jackson, R., J. G. Sliciano, Cinderella factors have a ball, Nature 394:830, 1998.

58. Preformed complex I is
not a dead end
Marker
17.32

59. Functions of eIF5 and eIF5B (eIF5 encourages association between 60S ribosome subunit and 48S complex)
eIF5B is homologous to prokaryotic factor IF2
Binds GTP
Uses GTP hydrolysis to promote its own dissociation from ribosome
Permits protein synthesis to begin
Stimulates association of 2 ribosomal subunits
Differs from IF2 as eIF5B cannot stimulate binding of initiating aminoacyl-tRNA to small ribosomal subunit
eIF5B works with eIF5

60. 17.3 Control of Initiation
Given the amount of control at the transcriptional and posttranscriptional levels, why control gene expression at translational level?
Major advantage = speed
New gene products can be produced quickly
Simply turn on translation of preexisting mRNA
Valuable in eukaryotes
Transcripts are relatively long
Take correspondingly long time to make
Most control of translation happens at the initiation step

61. Bacterial Translational Control
Most bacterial gene expression is controlled at transcription level
Majority of bacterial mRNA has a very short lifetime
Only 1 to 3 minutes
Allows bacteria to respond quickly to changing circumstances
Different cistrons on a polycistronic transcript can be translated better than others

62. Shifts in mRNA Secondary Structure
mRNA secondary structure can govern translation initiation
Replicase gene of the MS2 class of phages
Initiation codon is buried in secondary structure until ribosomes translating the coat gene open up the structure
Heat shock sigma factor, s32 of E. coli
Repressed by secondary structure that is relaxed by heating
Heat can cause an immediate unmasking of initiation codons and burst of synthesis

63. Proteins/mRNAs Induce mRNA Secondary Structure Shifts
Small RNAs with proteins can affect mRNA 2° structure to control translation initiation
Riboswitches can be used to control translation initiation via mRNA 2° structure
5’-untranslated region of E. coli thiM mRNA contain a riboswitch
This includes an aptamer that binds thiamine and its metabolite
Thiamine phosphate
Thiamine pyrophosphate (TTP)

64. Activation of mRNA Translation
When TPP abundant
Binds aptamer
Causes conformational shift in mRNA
Ties up Shine-Dalgarno in 2° structure
Shift hides the SD sequence from ribosomes
Inhibits translation of mRNA
Saves energy as thiM mRNA encodes an enzyme needed to produce more thiamine and TPP

65. Eukaryotic Translational Control
Eukaryotic mRNA lifetimes are relatively long, so there is more opportunity for translation control than in bacteria
eIF2 a-subunit is a favorite target for translation control
Heme-starved reticulocytes activate HCR (heme-controlled repressor)
Phosphorylates eIF2a
Inhibit initiation
Virus-infected cells have another kinase, DAI
Inhibits translation initiation

66. Repression of Translation by Phosphorylation

67. Phosphorylation of an eIF4E-Binding Protein
Insulin and a number of growth factors stimulate a pathway involving a protein kinase known as mTOR
mTOR kinase’s target protein
PHAS-1 (rat)
4E-BP1 (human)
Once phosphorylated by mTOR
This protein dissociates from eIF4E
Releases it to participate in active translation initiation

68. Stimulation of Translation by Phosphorylation of PHAS-1

69. Blockage of Translation Initiation by an miRNA
miRNA controls gene expression in two ways:
Degradation of mRNA when base-paired perfectly
Inhibition of protein production if not perfect match
Filipowicz et al conTranslation initiation that is cap-independent due to presence of an IRES, or a tethered initiation factor, is not affected by let-7 miRNA
This miRNA blocks binding of eIF4E to the cap of target mRNAs in the human cell

70. RNA
Degradation
80%
20%
10x
10x
P1573-

71. Blockage of Translation Initiation by an miRNA
The let-7 miRNA shifts the polysomal profile of target mRNAs in human cells toward smaller polysomes
This miRNA blocks translation initiation in human cells
Translation initiation that is cap-independent due to presence of an IRES, or a tethered initiation factor, is not affected by let-7 miRNA
This miRNA blocks binding of eIF4E to the cap of target mRNAs in the human cell

72. Fig
Collect and fractionate polysomes from cells transfected with RL-3xBulge gene and perform Northern blotting
Polysomes: A mRNA attached to several ribosomes (Ch. 19)
The more active translation initiation, the more ribosome will attach to the mRNA
Anti-miRNA inhibitor revert this effect

73. Fig

74. Blockage of Translation Initiation by an miRNA
Lin-4 miRNA does not alter the polysome profile of its target mRNA in C. elegans and does not appear to block translation initiation.
Different miRNAs have different modes-of-action
miRNAs work differently in different organisms
Or both