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Home , EIF2, EIF4A, EIF4G, Eukaryotic translation, Peptidyl transferase, Start codon

Basics II – Extension over BSc tRNA binding sites in

E-, P-, and A-site in the structural models shown with tRNA bound to it.

Alberts, Molecular of the 5/e © 2008 Wiley-VCH, Fig 6.64 Large subunit

Compare to previous slide of the 50S sub- unit of the .

Here, are in blue, RNA is in orange, and the in red.

Note that the proteins are looking like glued to the surface of the rRNA

figure from: dujs.dartmouth.edu/winter- 2010/nobel-prize-in-chemistry-2009-unraveling- the-ribosome%E2%80%99s-secrets Elongation I

youtube.com/watch?v=TfYf_rPWUdY youtube.com/watch?v=kmrUzDYAmEI

Initiation will be covered in much detail in the following  not included here

Alberts, of the Cell 5/e © 2008 Wiley-VCH, Fig 6.66 Elongation I Version 2. Semester (:

Die Polypeptidketten-Verlängerung (Elongation) ist ein zyklischer Vorgang mit drei klar voneinander unterscheidbaren Schritten:

Schritt 1 – Bindung einer neuen tRNA in Position A. Ein Aminoacyl-tRNA-Molekül wird an die leere A-Stelle neben einer besetzten P-Stelle an das Ribosom gebunden. Dort bildet es Basenpaare mit den drei mRNA-Nukleotiden, die an der A-Stelle exponiert sind.

Schritt 2 – Bildung der Peptidbindung. Das Carboxy-Ende der Polypeptidkette von wird von dem an der P-Bindungsstelle liegenden tRNA-Molekül getrennt und über eine Peptidbindung an die Aminosäure gebunden, die an das tRNA-Molekül in der A-Stelle gebunden ist. Katalysiert wird diese Reaktion von der Peptidyltransferase. Diese enzymatische Aktivität wird durch einen Abschnitt des großen rRNA-Moleküls in der großen Ribosomen-Untereinheit vermittelt.

Schritt 3 – Translokation und Freisetzung der tRNA aus Position B. Schließlich wird die neue Peptidyl-tRNA von der A-Stelle in die P-Stelle verschoben. Dabei bewegt sich das Ribosom um genau drei Nukleotide auf der mRNA weiter. Dieser Schritt erfordert Energie und wird durch eine Folge von Konformationsänderungen in Gang gesetzt, die durch Hydrolyse eines GTP-Moleküls getrieben werden. Während des im dritten Schritt ablaufenden Translokationsvorgangs löst sich das im zweiten Schritt an der P-Stelle gebildete freie tRNA-Molekül vom Ribosom und kehrt in den cytoplasmatischen tRNA-Vorrat zurück. Daher ist am Ende des dritten Schritts die A-Stelle wieder frei und kann eine neue tRNA auf-nehmen, an welche die nächste Aminosäure gebunden ist. Damit beginnt der ganze Vorgang von neuem. Elongation II – Peptidyltransferase

peptidyl is a  an adenin-base of ribosomal RNA catalyzes bond formation  the catalytic site is located on the large subunit relevance for our view on molecular evolution  synthesis is still catalyzed by RNA – there are no ribosomal proteins near the actual catalytic site.  under lab conditions elongation is possible without protein components of the ribosome  there are still ribosomal proteins whose function is unknown

Bacterial peptidyltransferase - target of several types of (outdated; used for lab purposes)  (topical; in part experimental)  (alternative to penicillin) Elongation III ‘chemical‘ representation of formation

Beringer and Rodnina, Mol Cell 26, 311ff Abstract to historical paper on next slide The Structural Basis of Ribosome Activity in Peptide Bond Synthesis Poul Nissen, Jeffrey Hansen, Nenad Ban, Peter B. Moore, and Thomas A. Steitz* Using the atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with two substrate analogs, we establish that the ribosome is a ribozyme and address the catalytic properties of its all-RNA active site. Both substrate analogs are contacted exclusively by conserved ribosomal RNA (rRNA) residues from domain V of 23S rRNA; there are no protein side-chain atoms closer than about 18 angstroms to the peptide bond being synthesized. The mechanism of peptide bond synthesis appears to resemble the reverse of the acylation step in proteases, with the base of A2486 (A2451 in ) playing the same general base role as -57 in chymotrypsin. The unusual pKa (where Ka is the acid dissociation constant) required for A2486 to perform this function may derive in part from its hydrogen bonding to G2482 (G2447 in E. coli), which also interacts with a buried phosphate that could stabilize unusual tautomers of these two bases. The polypeptide exit tunnel is largely formed by RNA but has significant contributions from proteins L4, L22, and L39e, and its exit is encircled by proteins L19, L22, L23, L24, L29, and L31e.

Steitz was awarded the 2009 Nobel Prize in Science 289, 920-930 along with Venkatraman Ramakrishnan and Ada Yonath ’for studies of the structure and function of the ribosome’. Chemical structures of ribo- some substrates and analogs (A) The tetrahedral carbon intermediate produced during peptide bond formation; the tetrahedral carbon is indicated by an arrow. (B) The transition state analog formed by coupling the 3' OH of CCdA to the amino group of the O-methyl residue of via a phosphate group, CCdA-p-Puro.

(C) An N-amino-acylated mini-helix constructed to target the A- site. The sequence 5'-CCG GCG GGC UGG UUC AAA CCG GCC CGC CGG ACC-3' puromycin should form 13 base pairs. The construct is based on a mini-helix known to be a suitable substrate for amino-acylation by Tyr-tRNA synthetase. The 3' OH of its terminal C is coupled to the 5' OH of the N6-dimethyl A moiety of puromycin by a phosphodiester bond

Note: Work of R. Schroeder here at the MFPL strengthened the interpretation that the function of several classical antibiotics (which are mostly natural substances) is based on their structural similarity to peptidyltransferase intermediates From where you might know puromycin? Science 289, 920-930 Puromycin is an aminonucleoside , from Streptomyces alboniger, that causes prema- ture chain termination during translation. Part of the molecule resembles the 3' end of aminoacylated tRNA. It enters the A site and transfers to the growing chain, cau- sing the formation of a puromycylated nascent chain and premature chain release. The 3' position contains an amide linkage instead of the normal ester linkage of tRNA. That makes the molecule more resistant to hydrolysis and stops the ribosome. Puromycin is used in as selective agent. It is toxic to prokaryotic and eukaryotic cells. Resistance to puromycin is conferred by the Pac encoding a puromycin N-acetyl-transferase (PAC) that was found in a Streptomyces producer strain.

soluble in water (50 mg/ml); colorless; standard stock solution10 mg/ml; stable for one year as solution at -20 °C; recommended dose as a selection agent 1-10 μg/ml, can be toxic to eukaryotic cells at concentrations as low as 1 μg/ml. Puromycin acts quickly and can kill up to 99% of nonresistant cells within 2 days. Binding sites of antibiotics on bacterial ribosomes Naturally occuring antibiotics are produced by fungi. Permanent concurrence  fungi for same nutrient sources Elongation IV – EFs and In vivo tRNA binding is facilitated / accelerated by elongation factors (EFs; Tu+G in , EF-

1+2 in ). Binding of EF-1 displaces the empty tRNA from the E-site under hydrolysis of GTP. The participa- tion of EF-1 greatly enhances the accuracy of codon-anticodon re- cognition. EF-2 facilitates the ~ synchronous movement of tRNAs from A+P sites to P+E, again using GTP hydrolysis as driving force. Under these conditions the rate of elongation is only 1-2 amino acids per second (~ 15 in prokaryotes). Termination I

https://www.youtube.com/watch?v=kmrUzDYAmEI&t=260s

Alberts, Molecular Biology of the Cell 5/e © 2008 Wiley-VCH, Fig 6.74 Termination II Die Termination verläuft bei Prokaryoten und Eukaryoten im Wesentlichen gleich. Sobald ein Stopcodon (UAA, UAG oder UGA) in die A-Stelle gelangt, kommt es zur Termination der Translation. Es gibt kein tRNA-Molekül, dessen Anticodon mit einem der Stop-Codons eine Basenpaarung eingehen könnte. Stattdessen besetzt einer von zwei Terminationsfaktoren (bei Eukaryonten eRF1) die A-Stelle des Ribosoms. Ein zweiter Terminationsfaktor (eRF3) erfüllt bei diesem Vorgang eine Hilfsfunktion. Die Bindung von eRF an die A-Stelle ändert die Aktivität der Peptidyltransferase so, dass diese ein Wassermolekül anstelle einer Aminosäure an die Peptidyl-tRNA anhängt. Dadurch wird das Carboxy-Ende der Polypeptidkette aus der Bindung an das tRNA-Molekül gelöst.

Da normalerweise die wachsende Polypeptidkette ausschliesslich durch diese Bindung mit dem Ribosom verknüpft ist, wird die fertige Proteinkette ins Cytoplasma entlassen. Anschließend setzt das Ribosom auch die mRNA und die tRNA der zuletzt eingebauten Aminosäure frei und zerfällt in seine beiden Untereinheiten.Diese können sich sogleich wieder an eine mRNA anlagern.

In eukaryotes, eRF1 recognizes all three termination codons, in procaryotes eRF1 recognizes UAA and UAG, eRF2 binds to UGA. high resolution animation of translation: youtube.com/watch?v=TfYf_rPWUdY Ribosome dynamics and tRNA movement by time- resolved electron cryomicroscopy Fischer N, Konevega AL, Wintermeyer W, Rodnina MV, Stark H Nature 466(#7304), 329-333, 2010 July 15 Dynamic mechanism of tRNA translocation during protein synthesis observing the classical A, P and E sites for tRNA binding as well as the hybrid A/P and P/E states during the transition. The authors hypothesize that the ribosome is a 'Brownian machine' that couples spontaneous, thermally-induced motions into directional movement. 1st ‘movie’* (20 frames GIF) showing 4 views onto a 70S procaryotic ribosome from top (upper row), from the solvent side of the 30S subunit (lower left) and onto 50S subunit and tRNAs (lower right). Note the coupling between global dynamics of the 30S subunit (yellow; left column), tRNA movement (right column), and local conformational changes of the 50S subunit (lower right). 2nd movie (11 frames GIF) showing 50S ribosomal subunit (semi-transparent grey) at larger resolution, indicating the 50S dynamics during translocation in molecular detail.

* homepage.univie.ac.at/ernst.muellner/transcon GIF on ribosome dynamics and tRNA movement Regulation of Initiation At multiple steps, including activity and availability . synthesis + degradation (eIF2, eIF4G) . (eIF2, eIF4E, eIF4E-BP) . low eIF4E abundance is limiting  proto-oncogene! (Dolznig lecture, summer) mRNA structure . cap accessibility . RNA secondary structure . uORFs . AUG context (Kozak-consensus sequence) RNA-RNA interactions

Protein-mRNA interactions (mRNA stability) mostly summer term . 5´UTR (ferritin, TMV, development, TOP mRNAs, S6-phosphoryl. . 3´UTR (poly-A tail, AREs, lox-mRNA, TfR, development) . co-operation between 5´and 3´UTR mRNA-localization mostly summer term . mainly in development and neuron function Roles of translation factors, complexes and regulatory proteins I Eukaryotic initiation factors

• eIF1 promotes binding of eIF2•GTP•Met–tRNAMet (methionyl-transfer RNA) and messenger RNA (mRNA) to the small (40S) ribosomal subunit. • eIF1A: promotes the binding of eIF2•GTP•Met–tRNAMet and mRNA to 40S. ■eIF2: binds GTP and Met–tRNAMet to form a ternary complex; delivers Met–tRNAMet to 40S. ■eIF2α:the regulatory subunit of eIF2; phosphorylated on Ser51 by general control non- derepressible 2 ().  ↓ inconsistent terminology ■eIF2B:guaninine exchange factor (GEF) for eIF2. • eIF3: promotes the binding of eIF2•GTP•Met–tRNAMet and mRNA to 40S. • eIF4A: an RNA and ATPase; a component of eIF4F. • eIF4B: a stimulator of eIF4A. ■eIF4E:7-methyl-GTP (m7GTP) cap-binding protein; a component of eIF4F. inconsistent terminology • eIF4F: the cap-binding complex, which consists of eIF4A, eIF4E and IF4G, promotes mRNA binding to the 43S complex.  inconsistent terminology ■eIF4G:the scaffolding component of eIF4F. eIF4G binds to eIF4E, eIF4A, poly(A)-binding protein (PABP), eIF3, and MAPK-interacting serine/ kinase 1 and 2 (Mnk1+Mnk2). • eIF5: the GTPase-activating protein (GAP) for eIF2. • eIF5B: a GTPase; promotes ribosome-subunit joining.

Klann and Dever, Nat Revs Neurosci 5(12), 931-942, (2004) Roles of translation factors, complexes and regulatory proteins II Other proteins and complexes

■ PABP: poly(A)-binding protein,which also binds to eIF4G. ■ 4E-BP: a translation-regulatory protein; binds to eIF4E and inhibits eIF4F formation. • CPEB: an RNA-binding protein; recognizes the cytoplasmic element (CPE) in the 3’ (UTR) of target mRNAs. • Maskin: a protein that binds to both CPEB and eIF4E, and blocks translation initiation by preventing eIF4F formation on the masked (repressed) mRNA. ■ 43S pre-initiation complex: a translation complex that consists of the 40S subunit, eIF2•GTP•Met–tRNAMet, eIF1, eIF1A, eIF3 and possibly eIF5. ■ 48S pre-initiation complex: a translation complex that consists of the 43S complex and mRNA.

Klann and Dever, Nature Reviews Neuroscience 5(12), 931-942, (2004) NOTE: Terminology did not really ‘improve’ since the 2004 review …

Jackson, R.J., Hellen, C.U., and Pestova, T.V. (2010). The mechanism of initiation and principles of its regulation. Nat Rev Mol Cell Biol 11, 113-127. NOTE: terminology did not really improve since the 2004 review …

… but the corresponding 2010 NatRevMolCellBiol version is cool, more later The mechanism of eukaryotic translation initiation and principles of its regulation Jackson, R.J., Hellen, C.U., and Pestova, T.V. (2010). Nat Rev Mol Cell Biol 11, 113-127 Protein synthesis is principally regulated at the initiation stage (rather than during elongation or termination), allowing rapid, reversible and spatial control of . Progress over recent years in determining the structures and activities of initiation factors, and in mapping their interactions in ribosomal initiation complexes, have advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studying the regulation of translation initiation by mechanisms that include the modulation of initiation factor activity (which affects almost all scanning-dependent initiation) and through sequence-specific RNA-binding proteins and (which affect individual mRNAs). Pathway of translation initiation in eukaryotes

A binary complex of eukaryotic Klann and Dever, Nature Reviews translation initiation factor 2 (eIF2) Neuroscience 5(12), 931-942, (2004) and GTP binds to methionyl-transfer RNA (Met–tRNAMet), and this ter- nary complex associates with the 40S ribosomal subunit. The asso- ciation of additional factors, such as eIF3 and eIF1A (1A), with the 40S subunit promotes ternary complex binding and generates a 43S pre- initiation complex. The cap-binding complex, which consists of eIF4E (4E), eIF4G and eIF4A (4A), binds to the 7-methyl-GTP (m7GTP) cap structure at the 5’end of a messenger RNA (mRNA). eIF4G also binds to the poly(A)-binding protein (PABP), thereby bridging the 5’ and 3’ ends of the mRNA. This mRNA circularization and the ATP-dependent helicase activity of eIF4A are thought to promote the binding of the 43S pre-initiation complex to the mRNA, which produces a 48S pre-initiation complex. Following scanning of the ribosome to the AUG , GTP is hydrolysed by eIF2, which triggers the dissociation of factors from the 48S complex and allows the eIF5B- and GTP-dependent binding of the large, 60S ribosomal subunit. Although the precise timing and requirements for the release of factors from the pre-initiation complexes are not clear, the 80S product of the pathway is competent for translation elongation and protein synthesis. eIF4G is THE adapter molecule … … to circularize mRNA into a translation-competent configuration

Translational control and signaling  H Dolznig

TIBS 24, 398-403 (1999), Dever-TE Figure legend to previous: eIF4G as *the* adapter

Recruitment of mRNA to the ribosome requires multiple protein–protein interactions involving eIF4G. The m7G cap-binding protein eIF4E interacts with a small region near the center of eIF4G, while eIF4A, an RNA helicase, binds to two regions in the C- terminal half of eIF4G. These three proteins comprise the cap-binding complex eIF4F. The polyA-binding protein (PABP) binds to a region near the N-terminus of eIF4G and, together with eIF4E, circularize mRNAs and promote translation of polyadenylated mRNAs. The multi-subunit eIF3 serves as a bridge between eIF4G and the ribosome. Regulation of translation is achieved by modifying and exploiting interactions with eIF4G. The MNK1 , which is activated by the mitogen-activated protein (MAPKs) ERK and p38, binds near the C-terminus of eIF4G where it can gain access to its substrate eIF4E. Phosphorylation of eIF4E increases its affinity for RNA. The binding of eIF4E to eIF4G is regulated by 4E-BP. Phosphorylation of 4E-BP, possibly by a MAPK, blocks its interaction with eIF4E and promotes translation.

As indicated by the scissors, several (and cellular proteases during !) block cap-dependent translation by cleaving eIF4G (polio-, coxsackie virus, rhino- virus, foot and mouth disease virus) or PABP () via virus-encoded proteases. During apoptosis similar cleavage processes can take place, in this case mediated by endogenous proteases. text modified from TIBS 24, 398-403 (1999), Dever-TE Regulation of erIF-4E activity Nat Rev Immunol 14, 361-376, June 2014 nature.com/nri/journal/v14/n6/pdf/nri3682.pdf

only upon inhibition of IRAK2 by IRAKM

Figure legend is from a review on innate immunity, therefore TLR etc. Compare to previous slide TLR or interleukin-1 receptor (IL-1R) engagement induces the phosphorylation of eIF4E in an IL-1R-associated kinase 2 (IRAK2)-dependent and MAPK-interacting protein kinase 1 (MNK1)- dependent or MNK2-dependent manner to stimulate the translation of a subset of mRNAs. TLR engagement also activates the mammalian target of rapamycin (mTOR) pathway, which leads to eIF4E-binding protein (4EBP) phosphorylation, thus releasing the cap-binding protein eIF4E to stimulate the translation of mRNAs with highly structured 5ʹ untranslated regions (5ʹ UTRs) Signaling pathways involved in  H Dolznig Regulation of mTOR by nutrients, growth factors, and stress

Growth factors activate mTORC1 through multiple path- ways. Black lines signify activating connections, whereas red lines signify inhibitory inputs between proteins. Dotted lines indicate connections between proteins that are not known to be direct.

Sengupta, et al., Mol. Cell 40, 310-322, (2010) Abstract to previous Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress

The large serine/threonine protein kinase mTOR regulates cellular and organismal homeostasis by coordinating anabolic and catabolic processes with nutrient, energy, and oxygen availability and growth factor signaling. Cells and experience a wide variety of insults that perturb the homeostatic systems governed by mTOR and therefore require appropriate stress responses to allow cells to continue to function. Stress can manifest from an excess or lack of upstream signals or as a result of genetic perturbations in upstream effectors of the pathway. mTOR nucleates two large protein complexes that are important nodes in the pathways that help buffer cells from stresses, and are implicated in the progression of stress-associated phenotypes and diseases, such as aging, tumorigenesis, and diabetes. This review focuses on the key components of the mTOR complex 1 (frequently called TORC1) pathway and on how various stresses impinge upon them.

Shomit Sengupta, Timothy R. Peterson, David M. Sabatini, Molecular Cell, Volume 40(2), 310-322, 22 October 2010 Translation initiation in eukaryotes – all combined

Jackson et al., Nat Rev Mol Cell Biol 11, 113-127, 2010 Translation initiation in eukaryotes – all combined

Jackson et al. Nat Rev Mol Cell Biol 11, 113-127, 2010 Pathway of translation initiation in eukaryotes – figure legend The canonical pathway of eukaryotic translation initiation is divided into eight stages (2–9). These stages follow the recycling of post-termination complexes (post-TCs; 1) to yield separated 40S and 60S ribosomal subunits, and result in the formation of an 80S ribosomal initiation complex, in which Met-tRNAMet is base paired with the initiation codon in the ribosomal P-site and which is competent to start the translation elongation stage. These stages are: eukaryotic initiation factor 2 (eIF2)–GTP–Met-tRNAMet ternary complex formation (2); formation of a 43S preinitiation complex comprising a 40S subunit, eIF1, eIF1A, eIF3, eIF2–GTP–Met-tRNAMet and probably eIF5 (3); mRNA , during which the mRNA cap-proximal region is unwound in an ATP-dependent manner by eIF4F with eIF4B (4); attachment of the 43S complex to this mRNA region (5); scanning of the 5′ UTR in a 5′ to 3′ direction by 43S complexes (6); recognition of the initiation codon and 48S initiation complex formation, which switches the scanning complex to a ‘closed’ conformation and leads to displacement of eIF1 to allow eIF5-mediated hydrolysis of eIF2-bound GTP and Pi release (7); joining of 60S subunits to 48S complexes and concomitant displacement of eIF2–GDP and other factors (eIF1, eIF3, eIF4B, eIF4F and eIF5) mediated by eIF5B (8); and GTP hydrolysis by eIF5B and release of eIF1A and GDP-bound eIF5B from assembled elongation- competent 80S ribosomes (9). Translation is a cyclical process, in which termination follows elongation and leads to recycling (1), which generates separated ribosomal subunits. The model omits potential ‘closed loop’ interactions involving poly(A)-binding protein (PABP), eukaryotic 3 (eRF3) and eIF4F during recycling, and the recycling of eIF2– GDP by eIF2B. Whether eRF3 is still present on ribosomes at the recycling stage is unknown. dnalc.org/view/15501-Translation-RNA-to-protein-3D-animation-with-basic-narration.html mRNA 3’ end for- mation and poly- adenylation I basic variant more in TransCon II

A poly(A) tail is added to the 3ʹ end of transcripts. The poly(A) signal and nearby U- rich or GU-rich downstream sequence elements are recognized by two multi- protein complexes — namely, cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulating factor (CSTF), respectively that promote endonucleolytic cleavage of the transcript. Poly(A) polymerase (PAP) catalyses the subsequent addition of a stretch of adenosines from the cleavage site.

Nat Rev Immunol14 | June 2014 | p 361ff; 361doi:10.1038/nri3682 Recycling of eIF2 by eIF2B and regulation by the eIF2 kinases The eukaryotic translation initiation factor 2 (eIF2)–GTP binary complex binds to methionyl-transfer RNA (Met–tRNAMet) and forms a ternary complex that then associates with the 40S ribosomal subunit. After start-codon recognition, GTP is hydrolysed by eIF2 and the binary eIF2-GDP complex is then released. The guanine nucleotide-exchange factor (GEF) eIF2B converts inactive eIF2-GDP to active eIF2-GTP, a process that is inhibited by phosphorylation (P) of the - subunit of eIF2 by one of the four known eIF2kinases. Phosphorylation of eIF2 converts eIF2 to a competitive inhibitor of eIF2B, and inhibition of eIF2B results in lowered levels of ternary complexes, which reduces general translation but increases translation of a specific class of messenger (mRNAs) with upstream open reading frames (uORFs).

Abbreviations: ATF4, activating factor 4; C/EBP, CCAAT / -binding protein; GCN, general control non- derepressible; HRI, haemregulated initiation factor 2kinase; m7G, 7-methyl-GTP; PERK, eIF2kinase 3; PKR, protein kinase- RNA regulated, -inducible double-stranded RNA Klann and Dever, Nature Reviews dependent. Neuroscience 5, 931-942, (2004) Protein kinases that regulate translation

Klann and Dever, Nat Revs Neuroscience 5, 931-942, (2004) Regulation of eIF2 activity by viral and ER stress

Nat Rev Immunol 14, 361-376, June 2014 nature.com/nri/journal/v14/n6/pdf/nri3682.pdf

Under normal conditions, eIF2 associates with a GTP molecule, a -initiator tRNA (Met- tRNAi) and the 40S ribosome to participate in translation initiation. After initiation, the GTP molecule is hydrolysed and eIF2 is released from the 40S ribosome. The GDP-associated eIF2 is then recycled by eIF2B into a GTP-associated eIF2 that can re-engage in translation. During viral infection or (ER) stress, eIF2 can be phosphorylated, which impairs its recycling by eIF2B, leading to translational inhibition of most mRNAs. Toll-like receptor (TLR) engagement under ER stress conditions leads to eIF2B stimulation, which in turn is able to efficiently recycle eIF2, even in its phosphorylated form, to maintain translation. Crucial initiation factors are composed of several subunits

Modular nature of the eIF2–eIF2B subunit interactions

The pentameric eIF2B can be divided into catalytic (2B, ) and regulatory (2B, , ) sub-complexes. It appears that eIF2B possesses the catalytic guanine-nucleotide-exchange activity, whereas the eIF2B, and  subunits are required for inhibition of eIF2B activity by eIF2 phosphorylated (P) on Ser51 of the  subunit. Three poly- (K) sequences near the N- terminus of eIF2 interact directly with a segment near the C-terminus of eIF2B. The points of contact between the regulatory and catalytic sub-complexes in eIF2B as well as the contacts between eIF2B and phosphorylated eIF2 are not known. The black circle TIBS 24, 398-403 (1999), Dever-TE on eIF2 represents the bound GDP nucleotide. Regulation of translational elongation ? I

SENSE? A number of signaling processes targets translational elongation NONSENSE? Translational initiation is THE rate-limiting step, isn't it? Relation between initiation rate elongation rate, ribosome density and rate of protein synthesis

Plotkin & Kudla, NatRevGenet 12, 32-42, (2011) Relationships between initiation rate, elongation rate, ribosome density and rate of protein synthesis The steady state rate of protein synthesis and density of ribosomes bound on an mRNA both depend on the rates of initiation and elongation. When elongation is the rate-limiting step in a gene’s translation (case A), the message will be covered as densely as possible by ribosomes and faster elongation will tend to increase the rate of protein synthesis. However, most endogenous are believed to be initiation limited (cases B, C and D), their transcripts are not completely covered by ribosomes. For two initiation-limited genes with the same initiation rate, the mRNA with faster elongation (e.g. higher codon adaptation to tRNA pools) will have a lower density of translating ribosomes (C versus B) but no greater rate of termination. Thus, when initiation is limiting, high codon adaptation should not be expected to increase the amount of protein that is produced per mRNA molecule (protein amounts are the same in B and C). A lower density of ribosomes can also occur when two initiation-limited genes have the same elongation rate, but one has a slower initiation rate (D versus C). In this case, the amount of protein that is produced will be lower for the mRNA that has the slower initiation rate (D). The extent to which variation in ribosome densities arises from variation in initiation versus elongation rates remains to be determined. In all cases shown here, as is true for most endogenous genes, the gene’s mRNA does not account for a substantial proportion of total cellular mRNA, so that the rates of initiation and elongation do not substantially alter the pool of free ribosomes Plotkin + Kudla, NatRevGenet 12, 32-42, (2011) special case – transgene expression

Plotkin + Kudla, NatRevGenet 12, 32-42, (2011) Regulation of translational elongation II

Eur J Bioch Vol 269, 5360pp, Browne and Proud

Translocation is catalyzed by eEF-2 coupled to GTP hydrolysis. In the process of translocation the ribosome is moved along the mRNA such that the next codon of the mRNA resides under the A site. Following translocation, eEF-2 is released from the ribosome. The cycle can now begin again. The ability of eEF-2 to carry out translocation is regulated by the state of phosphorylation of the , when phosphorylated the enzyme is inhibited. Phosphorylation of eEF-2 is catalyzed by the enzyme eEF2 kinase (eEF2K). Regulation of eEF2K activity is normally under the control of and Ca2+ fluxes / calmodulin. The Ca2+-mediated effects are the result of calmodulin interaction with eEF2K. eEF2K itself is also regulated by phosphorylation. !! eIF-2 + GTP  eEF-2 + GTP !! Regulation of translational elongation IIIa Inhibition of eEF2 and elongation by energy demand and other stimuli In response to activation of NMDA receptors or certain G-protein coupled receptors (GPCRs), intracellular Ca2+ levels rise, activating eEF2 kinase and leading to phosphorylation and inactivation of eEF2. Activation of adenylate cyclase, either by -adrenergic agonists or by forskolin, increases cAMP levels and activates cyclic AMP-dependent protein kinase (PKA). Ser500 phospho- This phosphorylates eEF2 kinase, activating it rylation activates Eur. J. Bioch. and leading to phosphorylation and inactivation eEF2 kinase (269), 5360pp, of eEF2. Modest depletion of cellular ATP (which Browne and Proud causes AMP levels to rise), or the direct activation of the AMP-activated protein kinase by AICA riboside, leads to increased phospho- rylation of eEF2, probably also through acti- vation of eEF2 kinase although the molecular mechanisms involved here are less clear. NMDAR, N-methyl-D-aspartate receptor; GPCR, G- protein coupled receptor; b-AR, b-adrenergic receptor; AC, adenylate cyclase; IP3, inositol trisphosphate. Regulation of translational elongation IIIb Activation of eEF2 by insulin, GPCR agonists and other stimuli. Insulin and IGF1 activate p70S6k (S6K1) via signalling events dependent upon mTOR (which is inhibited by rapamycin, shown). S6K1 phosphorylates eEF2 kinase at Ser366, and this inactivates eEF2 kinase, contributing to the dephosphorylation of eEF2. IGF1 also increases the phosphorylation of Ser359, a site which also inhibits eEF2 kinase activity. The kinase involved here is unknown, but phosphorylation is inhibited by rapamycin. Agents that activate the MEK/ERK pathway lead to activation of p90RSK1, which also phosphorylates Ser366 and inactivates eEF2 kinase. Such stimuli include the indicated GPCR agonists, which have been shown to decrease eEF2 phosphorylation in cardiomyocytes (see text). phosphorylation PD98059 and U0126 inhibit MEK activation, and block at the effects of these agents on eEF2 phosphorylation. inhibits Anisomycin stimulates several stress-activated protein eEF2 kinase kinase cascades, as indicated. Use of the p38 MAP kinase (SAPK2a/b) inhibitor SB203580 indicates that this pathway regulates the phosphorylation of Ser359 and Ser377, although SAPK4 is probably also involved Eur J Bioch (269), 5360pp, in the case of Ser359. IR/IGFR, insulin and/or IGF1 Browne and Proud receptors.