Molecular Biology Fourth Edition

1
Molecular BiologyFourth 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
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-tR
  NA
• 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 gt90 , 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 GarenFormyl-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 phages

• R17
• f2
• 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

• 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 3HO-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

25
(No Transcript)
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 3end of the 16S rRNA

27
The best evidence

• 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
SD sequence 16S rRNA GUGUG CCUCC (2 pairing) NO
SD sequence 16S rRNA CCUCC GGAGG (5 pairing) OK
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 complexlabeled 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 5end

• 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
• Kozaks 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 -3 -2 -1 1 2 3
4
41
(No Transcript)
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
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. 17.22
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

• eIF4A
• 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 polyA-binding protein
• Interacting with these proteins lets eIF4G
  recruit 40S ribosomal subunits to mRNA and
  stimulate translation

53
Fig. 17.27
Inhibit capped cellular mRNA
Synergy effect

• Regulatory protein and miRNA bound to the 3UTR
  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 394830,
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
• Phosphorylates eIF2a
• 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 kinases 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. 17.46
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. 17.47
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