Transcript Control of translation

Protein Translation
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Assembly of 5’-cap complex
Annealing of ribosome
t-RNA decoded polypeptide elongation
Trafficking
Co-translational modification
– Sugars
– Fatty acids
– Chaperone mediated folding
Control of translation
• General Mechanisms
– Activity of GTPases
– Availability of translation factors
• Protein specific mechanisms
– mRNA structure
– Sequence specific binding proteins
Control points in translation
• Cap binding structure
– eIF2-GTP+tRNA (GTP exchange)
– eIF4E (sequestration)
• Elongation
– eEF2-GTP+tRNA (GTP affinity)
• Sequence-specific mechanisms
– 5’ UTR structure
– Initiation complex efficiency
– RNA binding proteins
eIF2 Regulation
• Met-tRNA carrier; general translation rate
– 0.5 eIF2a per ribosome
• eIF2a kinases block GDP-GTP exchange
– Strengthen binding of eIF2a and eIF2B
– Extremely efficient: 20-30% p-eIF2a chelates
majority of eIF2B
eIF2B
eIF2a
phospho
phos-eIF2a
eIF2a
eIF2a Kinases
• “Stress” activated proteins
– Metabolic stress
– Environmental stress
• Reduce general translation in unhealthy
conditions
– Hemin-Regulated Inhibitory Kinase (HRI)
– General Control of amiNo acid synth (GCN2)
– Protein Kinase dsRNA activated (PKR)
– PKR-like Endoplasmic Reticulum Kinase
(PERK)
Hemin-Regulated Inhibitor kinase
• Constitutively active in reticulocytes &
erythrocytes
• Inhibited by heme to allow translation in
RBC precursors
• Balance globin synthesis to heme
availability
• Generally suppress translation by RBC
Hemoglobin
Heme
HRI
eIF2a
globin
GCN2
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General control of amino acid synthesis
Sensor for unloaded tRNA, AA abundance
Phosphorylates eIF2a, reduces protein synth
Stimulates GCN4 translation
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5’ upstream open reading frames
Re-initiation at GCN4 start only without eIF2
Transcriptional activator of amino acid biosynthesis
Activation of GCN4 in anterior piriform cortex
stimulates foraging behavior in mammals
Translation
AUG
GCN4 mRNA
ORF
Active coding sequence
PKR
• dsRNA-activated Protein Kinase
– dsRNA binding exposes ATPase
– Triggers dimerization &
autophosphorylation
– dsRNA viruses
• Induces Ifg &NF-kB
• PERK (PKR ER-related kinase)
– ER-Stress dependent
– Slows translation in response to misfolding
PERK
eIF2a
Translation
Misfolded
proteins
Healthy proteins
eIF4
• 4E Binding Proteins
– eIF4E cap binding protein
– Compete with eIF4G
– Phosphorylated after growth factor
activation
• Release eIF4E
• Thr-37 & Thr-46 (PI-3K/mTOR)
• Ser-65 & Thr-70 (ERK/CaMK?)
– Dephosphorylated by PP2A
• Bind eIF4E
4EBP
eIF4E
eIF4
43S
Translation
eEF phosphorylation
• eEF1B is the eEF1a GEF
– Phosphorylation increases activity
• PKC
• MSK6
– Increases eEF1a recycle rate & availability of
tRNA
• eEF2
– Needs no GEF
– Phosphorylated in GTP binding domain
• CaMKIII = eEF2 Kinase
• PKA dependent activation of eEF2
– Blocks activity
eEF1B phosphorylation
• eEF1B phosphorylation increases
eEF1a recycle rate
• Increases tRNA availability
eEF2 phosphorylation
• eEF2 phosphorylation blocks GTP
binding
• Decreases ribosome procession
PI-3K cascade
• GFR mediated
activation of PI3K
• Generation of PIP3
• PH binding
– PKB/Akt
– PDK1
• mTOR
• Translational
Machinery
PI3K targets in translational control
• 4EBP1
– Releases eIF4E to promote initiation
• eIF4E
– Facilitates binding to eIF4G
• eEF2 Kinase
– Blocks calmodulin binding
– Reduces phosphorylation of eEF2B
• p70S6 Kinase
– Increases 5’-TOP translation
Specific Targeting by S6
phosphorylation
• 5’ terminal oligopyrimidine (CU) structure
• S6 protein of 40S subunit
– Phoshporylation increases
affinity for 5’TOP
• Ribosomal proteins
• eIFs, eEFs
Regulation of Termination
• Stop codon recognition depends on
context
• E coli RF2
– In-frame, premature UGA stop
– Low RF2 gives 1-base frameshift
readthrough
– RF2 translationally autoregulated
• RF association with eIF4
Poly(A) binding protein
• Translation efficiency
– In vivo, (competitive) using electroporation
• 5x faster with poly(A)
• 5x faster with 7mG
• 250-10,000x faster with poly(A) and 7mG
– Not in reconstituted systems
• Kessler & Sachs
– Pab1
• eIF4G binding
• poly(A) binding
Poly(A) binding protein
• Pab1:eIF4G association
– Loop formation, steric facilitation
• 3’UTR
– Conformational facilitation
• No apparent change in IP complexes
– Inhibition of inhibitors
Evaluation of translational efficiency
• Comparison of protein and mRNA
– RT-PCR/PCR/Northern Blot
– ELISA/Western Blot
• Polysome profiles
Transcriptional
mRNA
protein
– Sedimentation rate by HPLC
Faster sedimenting
Heavier
Translational
5’ UTR structure control
• 50-70 nt; longer is better
• Scanning model
• Upstream open reading
frame
• Stem-loop structures
20 structure of HCV RNA
– Self-complimentary
sequences
• Internal Ribosome Entry
Site (IRES)
5’
Residue 330
mRNA Binding Elements
• Iron response element: block 40S
binding
• 5’ TOP: promote 40S binding
• Bruno: spatial repression of oskar by
eIF4G competition
• Micro RNA
Iron Response Element
• Stereotypical hairpin-loop
• Iron Response Protein
– Low iron allows binding
• 5’ block 40S binding
– eg ferritin iron buffer
• 3’ shield vs nuclease
– eg transferrin receptor to
import Fe
• Fe-IRP is part of the
Kreb’s cycle
Developmental regulation by oskar
• Little transcription early in development
• Oskar expression defines the posterior
pole of flies
– Anatomical axes defined during oogenesis
– Propagated by subcellular localization
• Bruno suppresses oskar translation
– Begins phenotypic specialization
Single cell
Multi-cell
Bru1 localization in
zebrafish embryo
(Hashimoto et al.
2006)
Summary
• Regulatory elements in untranslated
regions of mRNA
– Analogous to promoter/enhancer elements
of DNA
• General translational efficiency controls
– Metabolic status
– Growth controls
• Mechanisms
– GTP turnover
– Co-factor availability