Transcript Inquiry into Life Twelfth Edition

Lecture PowerPoint to accompany
Molecular Biology
Fourth Edition
Robert F. Weaver
Chapter 17
The Mechanism of
Translation I: Initiation
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
17-2
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
17-3
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
17-4
Aminoacyl-tRNA Synthetase
Activity
17-5
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
17-6
Ribosomal Subunit Exchange
17-7
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
17-8
First Codon and the First
Aminoacyl-tRNA
• Prokaryotic initiation codon is:
– Usually AUG
– Can be GUG
– Rarely UUG
• Initiating aminoacyl-tRNA is N-formylmethionyl-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
17-9
N-Formyl-Methionine
17-10
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
17-11
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
17-12
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
17-13
30S Initiation Complex
The complete 30S initiation complex
contains one each:
– 30S ribosomal subunit
– mRNA
– fMet-tRNA
– GTP
– Factors IF1, IF2, IF3
17-14
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
17-15
Bacterial Translation Initiation
1. IF1 influences dissociation of
70S ribosome to 50S and
30S
2. Binding IF3 to 30S, prevents
subunit reassociation
3. IF1, IF2, GTP bind alongside
IF3
4. Binding mRNA to fMet-tRNA
forming 30S initiation
complex
a. Can bind in either order
b. IF2 sponsors fMet-tRNA
c. IF3 sponsors mRNA
5. Binding of 50S with loss of
IF1 and IF3
6. IF2 dissociation and GTP
hydrolysis
17-16
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
17-17
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% cases ribosomal subunits bypass
1st AUG scanning for more favorable one
17-18
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
17-19
Scanning Model for Translation
Initiation
17-20
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
17-21
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
17-22
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
17-23
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
17-24
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
17-25
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
17-26
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
17-27
Prevention of Premature 40S60S 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
17-28
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
17-29
Principle of the Toeprint Assay
Source: Adapted from Jackson, R., J. G. Sliciano, Cinderella factors have a ball, Nature 394:830, 1998.
17-30
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
17-31
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
17-32
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
17-33
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
17-34
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)
17-35
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
17-36
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 (hemecontrolled repressor)
• Phosphorylates eIF2a
• Inhibit initiation
– Virus-infected cells have another kinase, DAI
– Phosphorylates eIF2a
– Inhibits translation initiation
17-37
Phosphorylation of an eIF4EBinding 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
17-38
Repression of Translation by
Phosphorylation
17-39
Stimulation of Translation by
Phosphorylation of PHAS-1
17-40
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
17-41
Stimulation by an mRNABinding 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
17-42
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
17-43