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