Eukaryotic Translation
Regulation
PTRM Class
February 8, 2011
Eukaryotic and Prokaryotic Translation
Differences
1. Translation and Transcription are uncoupled.
2. mRNAs are monocistronic.
3. mRNA has a 5’cap structure.
4. mRNA has a poly(A) tail (except Histone mRNA)
5. Ribosomes bind to the RNA at the 5’end (no Shine
Dalgarno sequence).
6. tRNAmeti is specific for initiation in eukaryotes it is not a
formylated tRNAmet
Similarities
1. The genetic code is nearly universal, applying to all species
on our planet. Protein synthesis begins with an AUG and
terminates with UGA, UAA, and UAG.
mRNA
(A)n 3'
Cap
Poly(A) tail
Secondary structures
IRES
Protein/RNA binding sites
uORF
miRNA target
Localization signals
AUG Kozak consensus sequence: GCC(A/G)CCAUGG
Fátima Gebauer & Matthias W. Hentze
Nature Reviews Molecular Cell Biology
5, 827-835 (2004)
eIF: Eukaryotic Initiation Factor
Steps of eukaryotic translation
initiation
Translational Control:
Mediated at the Level of Translation Initiation
The 40S ribosomal subunit is recruited to the 5’end of the mRNA through recognition
of the cap structure and interactions between translation initiation factors.
Eukaryotes
Gebauer & Hentze Nature Reviews
Molec. Cell Bio. 5, 827-835 (2004)
Prokaryotes
Base pairing between the Shine Dalgarno sequence and the 3´ end of 16S rRNA
facilitates translation initiation.
Voet and Voet, Figure 30-42
Scanning model of Cap-dependent Translation Initiation
1.
2.
Formation of the ternary
complex
Formation of the 43S preinitiation complex
3.
Recruitment of the 43S to
the 5’end of the mRNA
4.
40S ribosome scans down to
the start codon, positioning
met-tRNAi into the P-site.
5.
The 60S subunit joins,
facilitated by eIF5B
E P A
E P A
Fátima Gebauer & Matthias W. Hentze (2004)
Nature Reviews Molecular Cell Biology 5, 827-835
Role of eIF1 and eIF1A in initiation
IF3
IF1
eIF1 is proposed to monitor base
pairing between mRNA and MettRNAiMet
eIF1A: maintains the fidelity of
start codon recognition, likely
through an interaction with eIF5
and by stabilizing the open,
scanning-competent 40S
conformation until the start
codon is identified.
Also, eIF1A may bind to the A
site, blocking initiator tRNA from
binding in this position.
eIF1 and eIF1A enhance ternary
complex loading by altering the
conformation of the 40S subunit.
E
IF2
Fátima Gebauer & Matthias W. Hentze
Nature Reviews Molecular Cell Biology
5, 827-835 (2004)
P A
eIF1A plays an additional role by
stabilizing ternary complex once
it is bound.
Model for the Functions of eIF1 and eIF1A in Eukaryotic Tran
L. A. Passmore et al., Mol Cell 26, 41 (Apr 13, 2007).
Solvent surface
Closed
Open
n= neck
b= beak
sh= shoulder
lf= left foot
rf=right foot
pt=platform
40S-eIF1-eIF1A structure
represents an open,
scanning-competent form
of the initiation complex.
Intersubunit
surface
1.A new connection between
the head and shoulder on the
solvent side.
2.The ‘‘latch’’ of the mRNA
entry channel is closed in
empty 40S but is not visible in
the 40S-eIF1-eIF1A structure.
This latch is a noncovalent
interaction between rRNA
elements in the body
Clamping down of the latch following start codon
recognition and eIF1 release
The density for the latch appears more pronounced in 40S-eIF1A,
suggesting that the h18-h34 connection may be stronger when eIF1A is
bound. Because eIF1 dissociates after start codon recognition,
perhaps eIF1A, when present without eIF1, helps the 40S subunit to clamp
down on the mRNA, holding it in position in preparation for 60S subunit
joining.
L. A. Passmore et al., Mol Cell 26, 41 (Apr 13, 2007).
Recycling of the ternary complex requires eIF2B
Fátima Gebauer & Matthias W. Hentze
Nature Reviews Molecular Cell Biology
5, 827-835 (2004)
The ternary complex binds the 40S subunit and is
recruited to the 5’end of the mRNA
3
E P A
Fátima Gebauer & Matthias W. Hentze
Nature Reviews Molecular Cell Biology
5, 827-835 (2004)
7-methylguanosine cap structure
eIF4G is a scaffolding protein
Fátima Gebauer & Matthias W. Hentze
Nature Reviews Molecular Cell Biology
5, 827-835 (2004)
mRNA Circularizes Through eIF4G Binding to
eIF4E and PABP
http://departments.oxy.edu/biology/Stillman/bi221/091300/091300_lecture_figures.htm
eIF4E binding protein (4E-BP) Sequesters eIF4E
from eIF4F complex.
Apoptosis
Insulin
AA (Leucine)
Cell Proliferation
The 40S Ribosomal Subunit scans 5’ to 3’ to the
initiating AUG
PABP
4A
4G
3
1A
1
4E cap
E
5
P
A
Start site selection
•Proximity to the 5’end
•Kozak consensus sequence GCC(A/G)CCAUGG
•Scanning is an ATP dependent process
•eIF1 and 1A are involved in scanning, eIF4A is an RNA dependent helicase;
•eIF1 monitors the fidelity of codon-anticodon interaction
• eIF5 promotes GTP hydrolysis of the ternary complex following recognition
of the start codon.
Fátima Gebauer & Matthias W. Hentze
Nature Reviews Molecular Cell Biology
5, 827-835 (2004)
eIF5B Facilitates Ribosomal Subunit Joining
3
1A
5
1
E
P
A
eIF5B
E
P
A
Fátima Gebauer & Matthias W. Hentze
Nature Reviews Molecular Cell Biology
5, 827-835 (2004)
Electronmicrograph of a polysome: one mRNA strand (faint
horizontal line) with many individual ribosomes attached
(dark blobs). The newly synthesized polypeptide chains
(proteins) can be seen as irregularly shaped extensions from
the ribosomes:
http://bass.bio.uci.edu/~hudel/bs99a/lecture21/polysome.gif
Polysome Analysis
1.
2.
3.
4.
5.
6.
Freeze ribosomes onto the
mRNA with cyclohexamide
Load cell lysates onto a sucrose
gradient.
Use centrifugal force to separate
mRNAs based on their
translational efficiency
Fractionate the gradient.
Isolate total RNA from each
fraction.
Perform northern analysis to
look at translation of specific
mRNAs
Polysomes
Actin
Northern
Toe Print Assay
Pisarev et al. (2007) Methods in Enzymology 430: 147-177
What are the limits and advantages of polysome analysis and
Toe Print assays?
What are the limits and advantages of polysome analysis and
Toe Print assays?
Toe prints don’t tell you what size the complex is: (40S or 80S)
Polysomes indicate the global translation rates, where as
Northern analysis can tell you the translation status of a
particular mRNA
Toe prints can indicate exactly where on the mRNA a complex is
positioned.
Translational control by elements in the 5’UTR
1. Leaky scanning
2. IRES
2. Structure and/or Protein binding (IRE/IRP)
3. uORF (GCN4)
Leaky scanning
•Permits a downstream AUG to be used for initiation in preference to, or in
addition to the first (5’ proximal) AUG.
•ORFs may be in the same or different reading frames.
•The primary determining feature of start codon selection is the context of
the AUG:
•Kozak consensus sequence: GCCA/GCCAUGG
Gaba et. al. (2001) EMBO J. 20:6453-6463.
Two Mechanism of Translation Initiation
Cap-dependent
IRES-dependent
HCV
FMDV
E. Martínez-Salas et. al.
Journal of General Virology
(2001), 82, 973-984.
Cap-Independent Translation Initiation
• Cellular
– Cellular stress: viral infection (PKR), apoptosis, hypoxia
– Normal cellular processes: M phase cell cycle (25% translation)
• Viral
– Genomes: Picornaviruses, Hepatitis C Virus, Cricket paralysis
virus
– Specific viral messages: HIV, Herpes virus (…. and the list is still
growing!)
Cellular IRESes: How do they recruit the 40S
subunit?
• Widely assumed that the Secondary and tertiary
Structure allow for interactions with translational
machinery (ITAF,eIFs, 40S)
• No common Structure
• Composed of multiple short modules
• Generally 150-300 nts long, exception: 9nt repeated
element.
Dicistronic reporter Assay:
A functional Assay for IRES activity
Cap-dependent
40S
5’ cap
60S
IRES-dependent
40S60S
Renilla Luciferase
IRES
40S
60S
Firefly Luciferase
60S
AAAAA
40S
Marla Hertz
Ribosomes enter internally
Chen and Sarnow (1995) Science 268:415-417
Examples of IRES elements
E. Martínez-Salas et. al.
Journal of General Virology
(2001), 82, 973-984.
IRESs that bind to the 40S subunit in the absence of
initiation factors induce a similar conformational
change in the 40S subunit as does eIF1A and eIF1
binding. Suggesting that this is a general feature of
translation initiation.
Opening of the exit tunnel
h16 and rps3 connection
mRNA entry latch
40S subunit (yellow).
C. M. Spahn et al., Science 291, 1959
HCV IRES (purple) bound to
a 40S subunit (yellow).
Protein synthesis in poliovirus-infected HeLa cells
0 1 2 3 4 5 6
35S-meth.
pulse
80
-
51
-
109
34
26
eIF4G-Western
-
hrs p.i
Translational control by elements in the 5’UTR
1. IRES
2. Structure and/or Protein binding (IRE/IRP)
3. uORF (GCN4)
IRE/IRP: Steric Hindrance
Iron is both essential and toxic so its levels must be
carefully regulated within the cell. (Excess iron promotes the
generation of reactive radicals, which in turn damage cells and tissues.)
Transferrin receptor: Iron uptake
Ferritin: required for iron storage
KOSTAS PANTOPOULOS Ann. N.Y. Acad. Sci. 1012: 1–13 (2004)
eIF2 Phosphorylation Reduces Ternary Complex
Starvation (GCN2)
Viral infection, apoptosis (PKR)
ER Stress (PERK)
Haemin-regulated inhibitor (HRI)
Fátima Gebauer & Matthias W. Hentze
Nature Reviews Molecular Cell Biology
5, 827-835 (2004)
GCN4: Encodes a transcription factor for amino acid biosynthesis
Non-starvation
Starvation
Shunting/Re-initiation
A mechanism of translation initiation in which
ribosomes bind to the mRNA in a cap-dependent
manner (or at the 5’end) but then "jump" over large
regions of the mRNA containing RNA secondary
structure, upstream AUGs and open reading frames to
"land" at an AUG or upstream of the initiator AUG.
•Shunting
•Re-initiation
•Shunting/re-initiation
•Termination dependent re-initiation
•stop-start
Shunting
Examples:
Adenovirus (late mRNAs)
Sendaivirus (Y mRNAs)
Papillomavirus (E1 mRNA)
Duck hepatitis B virus
mammalian mRNAs
Properties:
1.Cap (or other mechanism)
2.RNA secondary structure
3.Motifs in the donor region that tether
the 40S to the RNA (some).
4.Some have uORF and require
termination of translation
L. A. Ryabova and T. Hohn, Genes Dev 14 (7), 817 (2000).
Shunting
Ribosomes bind to the
5’end
40S scans down the
message.
Some, translate an uORF
and terminate at a stop
codon.
40S subunit resumes
scanning
The 40S subunit “shunts”
to a downstream
sequence.
Translation initiates at a
downstream coding region.
L. A. Ryabova and T. Hohn, Genes Dev 14 (7), 817 (2000).
How do we demonstrate that the ribosome is
really jumping over the secondary sequence
and not scanning through it?
How do we demonstrate that the ribosome is
really jumping over the secondary sequence
and not scanning through it?
CaMV Shunting
mRNA, with the
5’takeoff and
3’acceptor site
CAT
activity: ++++
2 cotransfected
mRNAs,
∆5’takeoff
element
-
2 cotransfected
mRNAs,
∆3’acceptor
element
-
2 co-transfected
mRNAs,
With the 5’takeoff
and 3’acceptor
element
+
Futterer et. al. (1993) Cell 73:789-802.
Thought Question
How would you experimentally distinguish
between and IRES and a ribosomal
shunting mechanism?
Signals in the 3’UTR
Localization
zip codes
Stability
AU-rich elements
protein binding (transferrin receptor)
Translational control
Poly(A) addition during early development
miRNA targets
Fátima Gebauer & Matthias W. Hentze
Nature Reviews Molecular Cell Biology
5, 827-835 (2004)
mRNA Localization
Development:
Bicoid localized to the anterior pole of the
Drosophila embryo.
Neurons: mRNA is localized to the extremities
(dendrites and axons) of neurons which
allows rapid translational responses
independent of transcription in the nucleus.
Secreted or cell surface proteins: SRP encoded
by the nascent peptide directs the translating
ribosome to the ER
Protein and mRNA localization in Neurons
•Elements in the 3’UTR are bound by transacting factors in the nucleus.
•mRNA is translationally silent during translocation and after arrival.
•Translation is initiated upon an appropriate activation signal.
http://www.zmnh.uni-hamburg.de/izkn/ag_kindler.de.html
siRNA and miRNA
miRNAs translationally silence mRNA, in a
message specific manner
1. miRNA genes are found as single or clustered
transcriptional units, expressed from intron regions of
protein-coding or non-coding genes or as independent
transcriptional units.
2. Transcribed by polymerase II
3. The nuclear endoribonuclease, Drosha, cleaves pri-miRNAs
to generate 60 nt hairpin-containing precursors.
4. These are exported to the cytoplasm by exportin-5
5 The cytoplasmic endoribonuclease Dicer rapidly cleaves premiRNAs to yield mature 21nt miRNA duplex.
6. The guide strand of the miRNA duplex associates with an
miRNA induced silencing complex (miRISC) while the
passenger strand is degraded. (it is thought that the strand
with the weakest thermodynamic stability at the 5’ end is
incorporated into the RISC complex.
Complex algorithms predict that 30% of mammalian genes
are regulated by miRNAs.
miRNAs are expressed at more than 2,000 copies/cell in a
cell type and tissue specific manner.
They modulate the activities of genes that control cell
growth and differentiation.
miRNA
siRNA
Standart and Jackson (2007) G&D 21:1975-1982
Translational repression without affecting mRNA abundance:
initiation or elongation.
Degradation of mRNA through the classic deadenylation and decapping
pathway.
Summary
Mechanisms of initiation:
Cap-dependent translation
Cap-independent translation: IRES
Regulation of initiation:
Global
eIF2 alpha phosphorylation
4E-BPs
Cleavage of eIF4G (poliovirus infection, apoptosis)
Message specific
IRES
GCN4
IRE/IRP
miRNA (microRNA)
Localization
Ribosome shunting
Questions?