Protein Translation
• 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
• eIF2 kinases block GDP-GTP exchange– Strengthen binding of eIF2 and eIF2B– Extremely efficient: 20-30% p-eIF2 chelates
majority of eIF2B
phos-eIF2
eIF2B
eIF2
eIF2
phospho
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
HRI eIF2a globinHeme
Hemoglobin
GCN2
• General control of amino acid synthesis• Sensor for unloaded tRNA, AA abundance• Phosphorylates eIF2a, reduces protein synth• Stimulates GCN4 translation
– 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
GCN4 mRNA
ORF Active coding sequenceAUG
Translation
PKR
• dsRNA-activated Protein Kinase– dsRNA binding exposes ATPase – Triggers dimerization &
autophosphorylation– dsRNA viruses
• Induces If &NF-B
• PERK (PKR ER-related kinase) – ER-Stress dependent– Slows translation in response to misfolding
Translation
Misfoldedproteins
PERK
eIF2 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
eIF4E eIF4 43S4EBP Translation
eEF phosphorylation
• eEF1B is the eEF1 GEF– Phosphorylation increases activity
• PKC• MSK6
– Increases eEF1 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– Sedimentation rate by HPLC
mRNAprotein
Transcriptional Translational
Faster sedimentingHeavier
5’ UTR structure control
• 50-70 nt; longer is better
• Scanning model
• Upstream open reading frame
• Stem-loop structures– Self-complimentary
sequences
• Internal Ribosome Entry Site (IRES)
20 structure of HCV RNA
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
Bru1 localization in zebrafish embryo(Hashimoto et al. 2006)
Single cell
Multi-cell
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