1. Molecular Biology Fifth Edition
Lecture PowerPoint to accompany
Molecular Biology Fifth Edition
Robert F. Weaver
Chapter 17
The Mechanism of Translation I: Initiation
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. Translation
Translation is the process by which ribosomes read the genetic message in mRNA and produce a protein product according to the message
Ribosomes are protein factories
Transfer RNAs (tRNAs) play an important role as adaptors that can bind and amino acid at one end and interact with the mRNA at the other end

3. 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

4. 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

5. 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

6. Aminoacyl-tRNA Synthetase Activity

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

8. Ribosomal Subunit Exchange

9. Formation of the 30S Initiation Complex
Once the ribosomomal subunits have been dissociated, the 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

10. 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

11. N-Formyl-Methionine

12. 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

13. 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

14. 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 but GTP is not hydrolyzed in the process

15. 30S Initiation Complex
The complete 30S initiation complex contains one each:
30S ribosomal subunit
mRNA
fMet-tRNA
GTP
Factors IF1, IF2, IF3

16. 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

17. Bacterial Translation Initiation
IF1 influences dissociation of 70S ribosome to 50S and 30S
Binding IF3 to 30S, prevents subunit reassociation
IF1, IF2, and 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

18. 17.2 Initiation in Eukaryotes
Basic comparison of initiation between eukaryotes and bacteria
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

19. 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 in -3 position
G in +4 position
5-10% of the time, most ribosomal subunits bypass 1st AUG scanning for a more favorable one

20. Translation With a Short ORF
Sometimes ribosomes can use a short upstream open reading frame:
Initiate at an upstream AUG
Translate a short Open Reading Frame (ORF)
Continue scanning
Reinitiate at a downstream AUG

21. Scanning Model for Translation Initiation

22. 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

23. 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

24. 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

25. 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

26. Function of eIF4A and eIF4B
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

27. Function of eIF4G
eIF4G is a scaffold 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

28. Viral Corruption of Translation
Initiation of cellular translation can be corrupted by the picornavirus, poliovirus
A viral protease cleaves off the N-terminal domain of eIF4G so that it can no longer recognize caps and capped cellular mRNA is no longer translated
This cleavage leaves a C-terminal domain called p100
The loss of cap-dependent host protein synthesis in poliovirus infected cells is due to competition by viral RNA for the limitng amount of p100

29. 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
They are antagonistic; eIF1 promotes scanning while eIF1A causes the scanning 40S subunit to pause and commit to initiating at the right codon

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

31. Functions of eIF5 and eIF5B
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

32. 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

33. 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

34. 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

35. Proteins/mRNAs Induce mRNA Secondary Structure Shifts
Small RNAs with proteins can affect mRNA secondary 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)

36. 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

37. 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

38. Phosphorylation of an eIF4E-Binding Protein
Insulin and a number of growth factors stimulate a pathway involving a protein kinase complex known as mTORC1
Target protein for mTOR kinase is a protein called 4E-BP1
Once phosphorylated by mTOR
This protein dissociates from eIF4E
Releases it to participate in active translation initiation

39. Phosphorylation of an eIF4E-Binding Protein
Another target protein for mTOR kinase is S6K1
Once phosphorylated by mTOR
This protein phosphorylates from eIF4B, which facilitates its association with eIF4A, stimulating initiation of translation
It also phosphorylates PDCD4, which leads to the destruction of PDCD4 and the initiation of translation as PDCD4 is an eIF4A inhibitor

40. Repression of Translation by Phosphorylation

41. Control of Translation Initiation by Maskin
In Xenopus oocytes, Maskin binds to eIF4E and to CPEB (cytoplasmic polyadenylation element binding-protein)
Maskin bound to eIF4E, cannot bind to eIF4G, translation is now inhibited
Upon activation of oocytes
CPEB is phosphorylated
Polyadenylation is stimulate
Maskin dissociates from eIF4E
When Maskin is no longer attached
eIF4E able to associate with eIF4G
Translation can initiate

42. Repression by an mRNA-Binding Protein
Ferritin mRNA translation is subject to induction by iron
Induction seems to work as follows:
Repressor protein (aconitase apoprotein) binds to stem loop iron response element (IRE)
Binding occurs near 5’-end of the 5’-UTR of the ferritin mRNA
Iron removes this repressor and allows mRNA translation to proceed

43. 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