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The initiation phase of protein synthesis in eukaryotes The final phase of protein synthesis - Translational Termination The structure of a human translation release factor (eRF1) and its resemblance to a tRNA molecule Translational Control Two Types of Translational Control Two Types of Translational Control 1] Global regulation Two Types of Translational Control 1] Global regulation 2] Message specific regulation Review of Eukaryotic mRNA translation • Initiation - Recruitment of the ribosome to the mRNA Recognition of the start codon • Elongation -Movement of the ribosome along the mRNA Decoding the mRNA into an extending polypeptide chain • Termination - Recognition of the stop codon Release of the ribosome and protein chain from the mRNA start stop 7mGpppN 5’UTR ORF 3’UTR AAAAAAAAAAA Review of Eukaryotic mRNA translation • Initiation - Recruitment of the ribosome to the mRNA Recognition of the start codon start stop 7mGpppN 5’UTR ORF 3’UTR AAAAAAAAAAA Initiation Recruitment of ribosome and the initiator methionyl tRNA to the start codon Initiation Recruitment of ribosome and the initiator methionyl tRNA to the start codon eIF1A eIF2 eIF2B eIF3 eIF4A eIF4B eIF4E eIF4G eIF5 eIF5B aminoacylated initiator methionyl tRNA (Met-tRNAiMet) 40S ribosomal subunit 60S ribosomal subunit GTP ATP Initiation Recruitment of ribosome and the initiator methionyl tRNA to the start codon eIF1A eIF2 eIF2B eIF3 eIF4A eIF4B eIF4E eIF4G eIF5 eIF5B aminoacylated initiator methionyl tRNA (Met-tRNAiMet) 40S ribosomal subunit 60S ribosomal subunit GTP ATP Five Steps1. 40S ribosomal subunit and tRNAiMet preparation 2. mRNA selection and preparation 3. 40S/ mRNA binding 4. scanning and AUG recognition 5. 60S ribosomal subunit joining Step 1: 40S ribosomal subunit and tRNAiMet preparation 60S 60S 3 40S eIF2•GTP•Met-tRNAiMet is called the Met Ternary Complex-TC 3 2 GTP 40S 1A 1A 1 Met Met Met-tRNAiMet 2 GDP GTP 2B GTP 2 GDP 3 1 2 GTP 1A 43S pre-initiation complex Formation of Ternary Complex an important regulatory node 60S 60S 3 40S eIF2•GTP•Met-tRNAiMet is called the Met Ternary Complex-TC 3 2 GTP 40S 1A 1A 1 Met Met Met-tRNAiMet 2 GDP GTP 2B GTP 2 GDP 3 1 2 GTP 1A 43S pre-initiation complex Step 2: mRNA selection and preparation • The mRNA is bound by eIF4F complex composed of eIF4E, eIF4G, eIF4A. • eIF4B binds to eIF4A and facilitates the helicase activity of 4A that is required to unwind secondary structures in the mRNA during scanning eIF4F 4E Cap 4G 4B 4A AUG 11 Step 2: mRNA selection and preparation eIF4E binding is an important regulatory node 4E Cap eIF4F 4G 4B 4A AUG 12 Step 3: 43S/ mRNA binding and scanning 43S mRNA interaction eIF3 in the 40S complex and eIF4G in the mRNA complex interact Met 3 1 2 GTP 1A 43S pre-initiation complex 4G 4E 4B 4A Cap 3 AUG 48S pre-initiation complex 4E 4G Cap 3 4B 4A Met 1 GTP 2 1A AUG Step 3: 43S/ mRNA binding and scanning Scanning • The 48S complex scans each codon in a 5’ to 3’ direction looking for an AUG. • The eIF4A helicase activity irons out RNA hairpins allowing the 40S complex to move. • multiple rounds of ATP hydrolysis are required to provide energy for the movement 4E Cap 4G 4B 4A GTP 3 Met 2 1A 1 AUG Step 4: AUG recognition and GTP hydrolysis AUG recognition • eIFs 1, 2 and 5 are required for this step. 5 Met 3 Cap 2 5 1 1A AUG GTP GTPase step and recycling of eIFs • eIF5 stimulates the GTPase activity of eIF2 (GTP hydrolysis to GDP) leading to loss of initiation factor binding. Met 1A Cap AUG 2 GDP 3 1 5 Step 5: 60S Joining A second G protein eIF5B promotes 60S subunit joining. Hydrolysis of GTP to GDP promotes 5B and eIF1A release. The elongation phase, where the protein is synthesized can now begin. 60S 5B GTP Met Cap AUG 1A GTP GDP Met AUG Cap 5B GDP 1A 16 Elongati on Measurement of Translation Reporter Assays Reporter luciferase B-gal GFP poly(A) Westerns Extract proteins Run on SDS-Page Probe with Antibody 35S incorporation Pulse-label 35S met or cys Take time points CPM incorporated (X103 ) 5 4 3 2 1 0 Measure incorporation by TCA prec. followed by scintillation 0 2 4 6 Time (mins) 8 10 Yeast Extract Polysome analysis Sucrose gradient Non-translating RNAs mRNA 80S Translation efficiency 260nm Spin Abs polyribosomes Collect fractions 60S mRNA 40S 40S subunit Top 1 2 60S subunit Fractions 3 4 5 6 7 8 Bottom 9 10 11 12 13 14 15 16 polyribosomes mRNAs 80S ribosome rRNA 28S rRNA 18S rRNA Efficiently translated mRNA Poorly translated mRNA Two Types of Translational Control 1] Global regulation 2] Message specific regulation 1] Global regulation 80S RNP P 0 min Condition A 120 min Condition B GAL-Dhh1p 1] Global regulation 80S RNP P 0 min Condition A 120 min Condition B GAL-Dhh1p Usually seen during conditions of stress or stimulation by hormone 1] Global regulation eIF-4E regulation Ternary Complex formation Step 3: 43S/ mRNA binding and scanning 43S mRNA interaction eIF3 in the 40S complex and eIF4G in the mRNA complex interact Met 3 1 2 GTP 1A 43S pre-initiation complex 4G 4E 4B 4A Cap 3 AUG 48S pre-initiation complex 4E 4G Cap 3 4B 4A Met 1 GTP 2 1A AUG Three Types of eIF4E regulation 1] Transcriptional 2] eIF4E phosphorylation 3]Binding of inhibitory proteins Transcriptional Regulation of eIF4E eIF4E is the least abundant initiation factor in cells Transcription increase several fold in fibroblast upon growth factor treatment Mechanism poorly understood, but involves c-Myc regulation. Links to cancer Phosphorylation of eIF4E eIF4E phosphorylated at a single site -Ser209 Phosphorylation increases translation rate Structure of eIF4E with m7GpppG analog Ser209 m7GpppG Phosphorylated Ser209 interacts with Lys159 to make a retractable salt bridge Clamping mRNA in the cap-binding slot Ser209 Lys159 12/2/99 932 Growth Factor, Hormones, Mitogens,8505_AR_28 and Cytokines 12/2/99 6:44 mediate PM Page 933 eIF4E phosphorylation Page 932 eIF4 INITIATION FACTORS GINGRAS, RAUGHT & SONENBERG TABLE 1 TABLE 1 Effect of various stimuli on eIF4E and 4E-BP1 phosphorylation Stimulus Cell type Adenovirus infection (early) Stimulus Cell type +b 227, 228 Insulin-like growth factor I Rat aortic smooth muscle + 371 + 208, 357 Swiss 3T3-L1 + 362, 367 + 235 Insulin-like growth factor II 358 Interleukin 1! CHO.K1 Interleukin 3 Myeloid progenitor Lipopolysaccharide B lymphocytes + 212 L-Pyrroline-5carboxylic acid Rabbit reticulocyte lysate + 372 Nerve growth factor PC12 + Platelet-derived growth factor NIH 3T3 Lung fibroblasts Swiss 3T3-L1; aortic SM + + 4E-BP1 HeLa, 293 -a Angiotensin II Vascular smooth muscle + Anisomycin Swiss 3T3, 293 CD4+ or Mature thymocytes CD8+ + Arsenite 293, CHO.K1 + Concanavalin A Peripheral blood mononuclear cells + DAMGOc CHO overexpressing "-receptor Epidermal growth factor Mammary epithelial cells P19 ! 177 359 + 360 + 274 + 361 Swiss 3T3, 3T3-L1, L1, PC12 + Gastrin AR4-2J tumor cells + 258 GMCSF + SLF Hematopoietic MO7e + 365 Heat shock Reuber hepatoma + 231 High glucose Isolated rat pancreatic islets + NIH 3T3 + 3T3-L1 + Insulin + 362–364 366 178, 193, 319 + 162, 362, 363 367 CHO + 199, 211 Skeletal muscle + 368, 369 Swiss 3T3; 32D; 293; CHO-IR + 227, 235, 238 370 (continued) Gingras, unpublished data). Nevertheless, these data argue that ERKs and p38 act as upstream effectors of eIF4E phosphorylation (Figure 5). It is unclear why eIF4E phosphorylation is not increased after treatment of cells with sorbitol, H2O2, or heat shock, which are potent activators of p38 MAP kinases (177). One hypothesis is that these stresses also decrease phosphorylation of the eIF4E- 933 (continued) References eIF4E Anti-CD3 Annu. Rev. Biochem. 1999.68:913-963. Downloaded from arjournals.annualreviews.org by CASE WESTERN RESERVE UNIVERSITY on 02/22/06. For personal use only. 6:44 PM Annu. Rev. Biochem. 1999.68:913-963. Downloaded from arjournals.annualreviews.org by CASE WESTERN RESERVE UNIVERSITY on 02/22/06. For personal use only. 8505_AR_28 eIF4E 4E-BP1 + References 177 + 238 + 194, 364 + 178, 193, 319 373 362, 367, 371 PHA + phorbol ester Human T cells + 170 Phorbol ester NIH 3T3, CHO, PBL, B cells + 178, 193, 199, 211, 212, 319, 359 3T3-L1, leukaemic T cells, retic. lysate Swiss 3T3 + 162, 359, 372 NIH 3T3 Swiss 3T3 CHO 3T3-L1 + + + U937, HeLa, ME180, BAEC, FS, HUVEC + Serum Tumor necrosis factor " + 235 + 178, 193, 256, 319 374 211 362, 367 + 177, 375 a-, No change in phosphorylation Increase in phosphorylation; blank space indicates not determined cAbbreviations: DAMGO, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin; GMCSF, granulocyte-macrophage colony stimulating factor; SLF, steel factor; PHA, phytohemagglutinin. b+, An excellent candidate for the eIF4E kinase is the MAP kinase-interacting protein kinase-1(MNK1; also called MAP kinase signal-integrating kinase). MNK1 was identified independently by two groups as a substrate for ERK1 and 8505_AR_28 12/3/99 12:50 PM Page 935 Signal Transduction Pathways Mediate eIF4E Phosphorylation 99.68:913-963. Downloaded from arjournals.annualreviews.org RESERVE UNIVERSITY on 02/22/06. For personal use only. eIF4 INITIATION FACTORS 935 Figure 5 Signaling pathways to eIF4E phosphorylation. The intracellular signaling pathways leading to eIF4E phosphorylation are diagrammed. AlsoMAP indicated are the MNK1= kinase-interacting protein kinase inhibitors (in italics) used to delineate the pathways. Both MNK1 and eIF4E interact with eIF4G, bringing the two proteins in close proximity, resulting in more efficient eIF4E phosphorylation. eIF4E interacting proteins eIF4E binding proteins exist in higher eukaryotes (4E-BP) Three found in mammals (4E-BP1, 4E-BP2, and 4E-BP3) Small (10-12 kDa), heat-stable, all contain eIF4E binding site Binding of 4E-BP does not alter eIF-4E affinity for the cap et aL 4E-BP proteins block eIF4E/eIF4G binding A.Haghighat - B --I, + H A-plY2n) i ;jS T -* ,43i P i J1-F F 1 Fig. 3. 4E-BP1 and p220 compete for binding to eIF-4E. (A) m7GDP-coupled agarose resin was inc ml) and recombinant murine eIF-4E in buffer A. The resin was washed in buffer A (3XHaghighat et al., then 1995 incub for 60 min at 4° C. The resin was washed in buffer A (3x 1 ml) and incubated further with buffer A Note: p220 old nomenclature for eIF4G 4E-BP proteins block eIF4E/eIF4G binding eIF4G peptide 4E-BP peptide GINGRAS, RAUGHT & SONENBERG Annu. Rev. Biochem. 1999.68:913-963. Downloaded from arjournals.annualreviews.org by CASE WESTERN RESERVE UNIVERSITY on 02/22/06. For personal use only. 938 Figure 6 The cocrystal structure of the 4E-BP1 and eIF4GII peptides bound to eIF4E. (A) The human 4E-BP1 peptide (orange) and the m7GDP cap analog (green) bind to opposing regions of eIF4E (blue); (B) The 4E-BP1 peptide binds to the dorsal Gingras et al., 1999 convex surface of eIF4E and adopts an extended L-shaped conformation; (C) the eIF4GII peptide (red) also adopts an extended L-shaped conformation when binding 8505_AR_28 12/3/99 1:32 PM Page 940 4E-BP binding to eIF4E is regulated by phosphorylation ownloaded from arjournals.annualreviews.org ERSITY on 02/22/06. For personal use only. 940 GINGRAS, RAUGHT & SONENBERG Figure 7 The binding of the 4E-BPs to eIF4E is regulated by phosphorylation. The 4E-BPs and eIF4Gs compete for a common binding site on eIF4E. Various stimuli increase the phosphorylation of the 4E-BPs. Hyperphosphorylated 4E-BPs have a relatively low affinity for eIF4E. Conversely, a decrease in 4E-BP phosphorylation increases the affinity of the 4E-BPs for eIF4E. Free eIF4E interacts with eIF4G to form the translationally active eIF4F complex. GPCR: G protein coupled receptor. Activation of eIF4E by two distinct yet re-enforcing mechanisms !"#$%&'()&*!& +,-.+,-/01(2!3 34/!'210&*0& 5)678&9,4',&4.&3-'6!3&-)& !"#$:&; 4)'/!(.!.&(<<4)420& <-/&2,!&=>&'(+&(*-?2&$@ !"#$%&4.&(1.-&('24A(2!3&*0& +,-.+,-/01(24-)&-<&42.& *4)34)B&+/-2!4)&2,/-?B,& '!11&B/-92,&.4B)(14)B ! 1] Global regulation eIF-4E regulation Ternary Complex formation Step 1: 40S ribosomal subunit and tRNAiMet preparation 60S 60S 3 40S eIF2•GTP•Met-tRNAiMet is called the Met Ternary Complex-TC 3 2 GTP 40S 1A 1A 1 Met Met Met-tRNAiMet 2 GDP GTP 2B GTP 2 GDP 3 1 2 GTP 1A 43S pre-initiation complex Amino acid depletion induces a rapid translational repression events in yeast 80S RNP P 0 min 120 min -Amino GAL-Dhh1p +Amino Acids Acids Despite this, some mRNA are upregulated Alan Hinnebusch and Colleagues 5.59:407-450. Downloaded from arjournals.annualreviews.org ESERVE UNIVERSITY on 02/22/06. For personal use only. ANRV253-MI59-18 ARI 4 August 2005 16:40 The Presence of uncharged tRNAs stimulates eIF2α phosphorylation Ternary Complex Levels High Non-starvation conditions Ternary Complex Levels Low Starvation conditions Figure 3 Model for GCN4 translational control. GCN4 mRNA and reinitiating ribosomes are depicted as in Step 1: 40S ribosomal subunit and tRNAiMet preparation 60S 60S 3 40S eIF2•GTP•Met-tRNAiMet is called the Met Ternary Complex-TC 3 2 GTP 40S 1A 1A 1 Met Met Met-tRNAiMet 2 GDP GTP 2B GTP 2 GDP 3 1 2 GTP 1A 43S pre-initiation complex NTD is sufficient for binding to a C-terminal segment of Gcn1p (approximately residues 2050–2400), and overexpressing either domain dissociates the native Gcn1p/Gcn2p complex and produces a Gcn− phenotype that Gcn1p function without affecting ribosome binding or Gcn20p binding by Gcn1p, thus showing that the Gcn1p-Gcn2p interaction is crucial. The extreme N-terminal (amino acids 1–672) and C-terminal (amino acids Annu. Rev. Microbiol. 2005.59:407-450. Downloaded from arjournals.annualreviews.org by CASE WESTERN RESERVE UNIVERSITY on 02/22/06. For personal use only. GCN2 kinase is a sensor for uncharged tRNA 422 Hinnebusch Amino acid depletion induces a rapid translational repression events in yeast 80S RNP P 0 min 120 min -Amino GAL-Dhh1p +Amino Acids Acids Despite this, some mRNA are upregulated Alan Hinnebusch and Colleagues !"#$%&'()*+#%+,%-%.'-#,"'+/)+(#%0-")('%)1-)% 2*,/(#3,%)(%45+#(%4"+3%6*7+"+*#"8 " #!% # 9 ! 9 $ !"#$ & :8#)1*,+,%(7%!"#$/%+,%'*;<=-)*3%-)%)1*%)'-#,=-)+(#-=%=*>*= & ?)%+,%'*;<=-)*3%@8%-#%*?0A! B+#-,*C%!"#A/ & 4")+>+)8%(7%!"#A/%+,%"(#)'(==*3%@8%<#"1-';*3%)2D4 icrobiol. 2005.59:407-450. Downloaded from arjournals.annualreviews.org WESTERN RESERVE UNIVERSITY on 02/22/06. For personal use only. Figure 3 Model for GCN4 translational control. GCN4 mRNA and reinitiating ribosomes are depicted as in Figure 2. The three subunits of eIF2 and the five subunits of eIF2B are listed in the left panel. Negative regulatory factors are depicted in red; positive effectors are depicted in green. Following translation of uORF1, ∼50% of the 40S ribosomes remain attached to the mRNA and resume scanning. Under nonstarvation conditions, the 40S subunit quickly rebinds the TC and reinitiates at uORF4 because the The GAAC response a translation control pathway that allows for survival when times are tough Amino acid levels reduced uncharged tRNA levels high Exogenous amino acid levels high charged tRNA levels high GCN2 non-ribosome bound, inactive eIF2α unphosphorylated GCN2 ribosome bound, active eIF2α phosphorylated Ternary complex levels reduced Ternary complex levels high Bulk translation decreases Translation rate high GCN4 mRNA translated Gcn4p dependent transcription of amino acid biosynthesis genes occurs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wo Types of Translational Control 1] Global regulation 2] Message specific regulation