eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining.

Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.

Human eukaryotic initiation factor 4G directly binds the 40S ribosomal subunit to promote efficient translation.

Messenger RNA (mRNA) recruitment to the 40S ribosomal subunit is mediated by eukaryotic initiation factor 4F (eIF4F). This complex includes three subunits: eIF4E (m7G cap-binding protein), eIF4A (DEAD-box helicase), and eIF4G. Mammalian eIF4G is a scaffold that coordinates the activities of eIF4E and eIF4A and provides a bridge to connect the mRNA and 40S ribosomal subunit through its interaction with eIF3. While the roles of many eIF4G binding domains are relatively clear, the precise function of RNA binding by eIF4G remains to be elucidated. In this work, we used an eIF4G-dependent translation assay to reveal that the RNA binding domain (eIF4G-RBD; amino acids 682-720) stimulates translation. This stimulating activity is observed when eIF4G is independently tethered to an internal region of the mRNA, suggesting that the eIF4G-RBD promotes translation by a mechanism that is independent of the m7G cap and mRNA tethering. Using a kinetic helicase assay, we show that the eIF4G-RBD has a minimal effect on eIF4A helicase activity, demonstrating that the eIF4G-RBD is not required to coordinate eIF4F-dependent duplex unwinding. Unexpectedly, native gel electrophoresis and fluorescence polarization assays reveal a previously unidentified direct interaction between eIF4G and the 40S subunit. Using binding assays, our data show that this 40S subunit interaction is separate from the previously characterized interaction between eIF4G and eIF3. Thus, our work reveals how eIF4F can bind to the 40S subunit using eIF3-dependent and eIF3-independent binding domains to promote translation initiation.

Monitoring RNA restructuring in a human cell-free extract reveals eIF4A-dependent and eIF4A-independent unwinding activity.

The canonical DEAD-box helicase, eukaryotic initiation factor (eIF) 4A, unwinds 5' UTR secondary structures to promote mRNA translation initiation. Growing evidence has indicated that other helicases, such as DHX29 and DDX3/ded1p, also function to promote the scanning of the 40S subunit on highly structured mRNAs. It is unknown how the relative contributions of eIF4A and other helicases regulate duplex unwinding on an mRNA to promote initiation. Here, we have adapted a real-time fluorescent duplex unwinding assay to monitor helicase activity precisely in the 5' UTR of a reporter mRNA that can be translated in a cell-free extract in parallel. We monitored the rate of 5' UTR-dependent duplex unwinding in the absence or presence of an eIF4A inhibitor (hippuristanol), a dominant negative eIF4A (eIF4A-R362Q), or a mutant eIF4E (eIF4E-W73L) that can bind the m7G cap but not eIF4G. Our experiments reveal that the duplex unwinding activity in the cell-free extract is roughly evenly split between eIF4A-dependent and eIF4A-independent mechanisms. Importantly, we show that the robust eIF4A-independent duplex unwinding is not sufficient for translation. We also show that the m7G cap structure, and not the poly(A) tail, is the primary mRNA modification responsible for promoting duplex unwinding in our cell-free extract system. Overall, the fluorescent duplex unwinding assay provides a precise method to investigate how eIF4A-dependent and eIF4A-independent helicase activity regulates translation initiation in cell-free extracts. We anticipate that potential small molecule inhibitors could be tested for helicase inhibition using this duplex unwinding assay.

Human eukaryotic initiation factor 4E (eIF4E) and the nucleotide-bound state of eIF4A regulate eIF4F binding to RNA.

During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.

5' Untranslated mRNA Regions Allow Bypass of Host Cell Translation Inhibition by Legionella pneumophila.

Legionella pneumophila grows within membrane-bound vacuoles in alveolar macrophages during human disease. Pathogen manipulation of the host cell is driven by bacterial proteins translocated through a type IV secretion system (T4SS). Although host protein synthesis during infection is arrested by the action of several of these translocated effectors, translation of a subset of host proteins predicted to restrict the pathogen is maintained. To identify the spectrum of host proteins selectively synthesized after L. pneumophila challenge, macrophages infected with the pathogen were allowed to incorporate the amino acid analog azidohomoalanine (AHA) during a 2-h time window, and newly synthesized macrophage proteins were isolated by orthogonal chemistry followed by mass spectrometry. Among the proteins isolated were interferon-stimulated genes as well as proteins translated from highly abundant transcripts. Surprisingly, a large number of the identified proteins were from low-abundance transcripts. These proteins were predicted to be among the most efficiently translated per unit transcript in the cell based on ribosome profiling data sets. To determine if high ribosome loading was a consequence of efficient translation initiation, the 5' untranslated regions (5' UTR) of transcripts having the highest and lowest predicted loading levels were inserted upstream of a reporter, and translation efficiency was determined in response to L. pneumophila challenge. The efficiency of reporter expression largely correlated with predicted ribosome loading and lack of secondary structure. Therefore, determinants in the 5' UTR allow selected host cell transcripts to overcome a pathogen-driven translation blockade.

A helicase-independent activity of eIF4A in promoting mRNA recruitment to the human ribosome.

In the scanning model of translation initiation, the decoding site and latch of the 40S subunit must open to allow the recruitment and migration of messenger RNA (mRNA); however, the precise molecular details for how initiation factors regulate mRNA accommodation into the decoding site have not yet been elucidated. Eukaryotic initiation factor (eIF) 3j is a subunit of eIF3 that binds to the mRNA entry channel and A-site of the 40S subunit. Previous studies have shown that a reduced affinity of eIF3j for the 43S preinitiation complex (PIC) occurs on eIF4F-dependent mRNA recruitment. Because eIF3j and mRNA bind anticooperatively to the 43S PIC, reduced eIF3j affinity likely reflects a state of full accommodation of mRNA into the decoding site. Here, we have used a fluorescence-based anisotropy assay to quantitatively determine how initiation components coordinate their activities to reduce the affinity of eIF3j during the recruitment of mRNA to the 43S PIC. Unexpectedly, we show that a full reduction in eIF3j affinity for the 43S PIC requires an ATP-dependent, but unwinding-independent, activity of eIF4A. This result suggests that in addition to its helicase activity, eIF4A uses the free energy of ATP binding and hydrolysis as a regulatory switch to control the conformation of the 43S PIC during mRNA recruitment. Therefore, our results define eIF4A as a universal initiation factor in cap-dependent translation initiation that functions beyond its role in RNA unwinding.

Cellular cap-binding protein, eIF4E, promotes picornavirus genome restructuring and translation.

Picornaviruses use internal ribosome entry sites (IRESs) to translate their genomes into protein. A typical feature of these IRESs is their ability to bind directly to the eukaryotic initiation factor (eIF) 4G component of the eIF4F cap-binding complex. Remarkably, the hepatitis A virus (HAV) IRES requires eIF4E for its translation, but no mechanism has been proposed to explain this. Here we demonstrate that eIF4E regulates HAV IRES-mediated translation by two distinct mechanisms. First, eIF4E binding to eIF4G generates a high-affinity binding conformation of the eIF4F complex for the IRES. Second, eIF4E binding to eIF4G strongly stimulates the rate of duplex unwinding by eIF4A on the IRES. Our data also reveal that eIF4E promotes eIF4F binding and increases the rate of restructuring of the poliovirus (PV) IRES. This provides a mechanism to explain why PV IRES-mediated translation is stimulated by eIF4E availability in nuclease-treated cell-free extracts. Using a PV replicon and purified virion RNA, we also show that eIF4E promotes the rate of eIF4G cleavage by the 2A protease. Finally, we show that cleavage of eIF4G by the poliovirus 2A protease generates a high-affinity IRES binding truncation of eIF4G that stimulates eIF4A duplex unwinding independently of eIF4E. Therefore, our data reveal how picornavirus IRESs use eIF4E-dependent and -independent mechanisms to promote their translation.

Molecular mechanism of poliovirus Sabin vaccine strain attenuation.

Recruitment of poliovirus (PV) RNA to the human ribosome requires the coordinated interaction of the viral internal ribosome entry site (IRES) and several host cellular initiation factors and IRES trans-acting factors (ITAFs). Attenuated PV Sabin strains contain point mutations in the PV IRES domain V (dV) that inhibit viral translation. Remarkably, attenuation is most apparent in cells of the central nervous system, but the molecular basis to explain this is poorly understood. The dV contains binding sites for eukaryotic initiation factor 4G (eIF4G) and polypyrimidine tract-binding protein (PTB). Impaired binding of these proteins to the mutant IRESs has been observed, but these effects have not been quantitated. We used a fluorescence anisotropy assay to reveal that the Sabin mutants reduce the equilibrium dissociation constants of eIF4G and PTB to the PV IRES by up to 6-fold. Using the most inhibitory Sabin 3 mutant, we used a real-time fluorescence helicase assay to show that the apparent affinity of an active eIF4G/4A/4B helicase complex for the IRES is reduced by 2.5-fold. The Sabin 3 mutant did not alter the maximum rate of eIF4A-dependent helicase activity, suggesting that this mutant primarily reduces the affinity, rather than activity, of the unwinding complex. To confirm this affinity model of attenuation, we show that eIF4G overexpression in HeLa cells overcomes the attenuation of a Sabin 3 mutant PV-luciferase replicon. Our study provides a quantitative framework for understanding the mechanism of PV Sabin attenuation and provides an explanation for the previously observed cell type-specific translational attenuation.

Mapping surface residues of eIF5A that are important for binding to the ribosome using alanine scanning mutagenesis.

The translation elongation factor eIF5A is conserved through evolution and is necessary to rescue the ribosome during translation elongation of polyproline-containing proteins. Although the site of eIF5A binding to the ribosome is known, no systematic analysis has been performed so far to determine the important residues on the surface of eIF5A required for ribosome binding. In this study, we used clustered charged-to-alanine mutagenesis and structural modeling to address this question. We generated four new mutants of yeast eIF5A: tif51A-4, tif51A-6, tif51A-7 and tif51A-11, and complementation analysis revealed that tif51A-4 and tif51A-7 could not sustain cell growth in a strain lacking wild-type eIF5A. Moreover, the allele tif51A-4 also displayed negative dominance over wild-type eIF5A. Both in vivo GST-pulldowns and in vitro fluorescence anisotropy demonstrated that eIF5A from mutant tif51A-7 exhibited an importantly reduced affinity for the ribosome, implicating the charged residues in cluster 7 as determinant features on the eIF5A surface for contacting the ribosome. Notably, modified eIF5A from mutant tif51A-4, despite exhibiting the most severe growth phenotype, did not abolish ribosome interactions as with mutant tif51A-7. Taking into account the modeling eIF5A + 80S + P-tRNA complex, our data suggest that interactions of eIF5A with ribosomal protein L1 are more important to stabilize the interaction with the ribosome as a whole than the contacts with P-tRNA. Finally, the ability of eIF5A from tif51A-4 to bind to the ribosome while potentially blocking physical interaction with P-tRNA could explain its dominant negative phenotype.

Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity.

Elevated eukaryotic initiation factor 4E (eIF4E) levels frequently occur in a variety of human cancers. Overexpression of eIF4E promotes cellular transformation by selectively increasing the translation of proliferative and prosurvival mRNAs. These mRNAs possess highly structured 5'-UTRs that impede ribosome recruitment and scanning, yet the mechanism for how eIF4E abundance elevates their translation is not easily explained by its cap-binding activity. Here, we show that eIF4E possesses an unexpected second function in translation initiation by strongly stimulating eukaryotic initiation factor 4A (eIF4A) helicase activity. Importantly, we demonstrate that this activity promotes mRNA restructuring in a manner that is independent of its cap-binding function. To explain these findings, we show that the eIF4E-binding site in eukaryotic initiation factor 4G (eIF4G) functions as an autoinhibitory domain to modulate its ability to stimulate eIF4A helicase activity. Binding of eIF4E counteracts this autoinhibition, enabling eIF4G to stimulate eIF4A helicase activity. Finally, we have successfully separated the two functions of eIF4E to show that its helicase promoting activity increases the rate of translation by a mechanism that is distinct from its cap-binding function. Based on our results, we propose that maintaining a connection between eIF4E and eIF4G throughout scanning provides a plausible mechanism to explain how eIF4E abundance selectively stimulates the translation of highly structured proliferation and tumor-promoting mRNAs.

Human eukaryotic initiation factor 2 (eIF2)-GTP-Met-tRNAi ternary complex and eIF3 stabilize the 43 S preinitiation complex.

The formation of a stable 43 S preinitiation complex (PIC) must occur to enable successful mRNA recruitment. However, the contributions of eIF1, eIF1A, eIF3, and the eIF2-GTP-Met-tRNAi ternary complex (TC) in stabilizing the 43 S PIC are poorly defined. We have reconstituted the human 43 S PIC and used fluorescence anisotropy to systematically measure the affinity of eIF1, eIF1A, and eIF3j in the presence of different combinations of 43 S PIC components. Our data reveal a complicated network of interactions that result in high affinity binding of all 43 S PIC components with the 40 S subunit. Human eIF1 and eIF1A bind cooperatively to the 40 S subunit, revealing an evolutionarily conserved interaction. Negative cooperativity is observed between the binding of eIF3j and the binding of eIF1, eIF1A, and TC with the 40 S subunit. To overcome this, eIF3 dramatically increases the affinity of eIF1 and eIF3j for the 40 S subunit. Recruitment of TC also increases the affinity of eIF1 for the 40 S subunit, but this interaction has an important indirect role in increasing the affinity of eIF1A for the 40 S subunit. Together, our data provide a more complete thermodynamic framework of the human 43 S PIC and reveal important interactions between its components to maintain its stability.

Human eukaryotic initiation factor 4G (eIF4G) protein binds to eIF3c, -d, and -e to promote mRNA recruitment to the ribosome.

Recruitment of mRNA to the 40S ribosomal subunit requires the coordinated interaction of a large number of translation initiation factors. In mammals, the direct interaction between eukaryotic initiation factor 4G (eIF4G) and eIF3 is thought to act as the molecular bridge between the mRNA cap-binding complex and the 40S subunit. A discrete ∼90 amino acid domain in eIF4G is responsible for binding to eIF3, but the identity of the eIF3 subunit(s) involved is less clear. The eIF3e subunit has been shown to directly bind eIF4G, but the potential role of other eIF3 subunits in stabilizing this interaction has not been investigated. It is also not clear if the eIF4A helicase plays a role in stabilizing the interaction between eIF4G and eIF3. Here, we have used a fluorescence anisotropy assay to demonstrate that eIF4G binds to eIF3 independently of eIF4A binding to the middle region of eIF4G. By using a site-specific cross-linking approach, we unexpectedly show that the eIF4G-binding surface in eIF3 is comprised of the -c, -d and -e subunits. Screening multiple cross-linker positions reveals that eIF4G contains two distinct eIF3-binding subdomains within the previously identified eIF3-binding domain. Finally, by employing an eIF4G-dependent translation assay, we establish that both of these subdomains are required for efficient mRNA recruitment to the ribosome and stimulate translation. Our study reveals unexpected complexity to the eIF3-eIF4G interaction that provides new insight into the regulation of mRNA recruitment to the human ribosome.

The human translation initiation multi-factor complex promotes methionyl-tRNAi binding to the 40S ribosomal subunit.

The delivery of Met-tRNA(i) to the 40S ribosomal subunit is thought to occur by way of a ternary complex (TC) comprising eIF2, GTP and Met-tRNA(i). We have generated from purified human proteins a stable multifactor complex (MFC) comprising eIF1, eIF2, eIF3 and eIF5, similar to the MFC reported in yeast and plants. A human MFC free of the ribosome also is detected in HeLa cells and rabbit reticulocytes, indicating that it exists in vivo. In vitro, the MFC-GTP binds Met-tRNA(i) and delivers the tRNA to the ribosome at the same rate as the TC. However, MFC-GDP shows a greatly reduced affinity to Met-tRNA(i) compared to that for eIF2-GDP, suggesting that MFC components may play a role in the release of eIF2-GDP from the ribosome following AUG recognition. Since an MFC-Met-tRNA(i) complex is detected in cell lysates, it may be responsible for Met-tRNA(i)-40S ribosome binding in vivo, possibly together with the TC. However, the MFC protein components also bind individually to 40S ribosomes, creating the possibility that Met-tRNA(i) might bind directly to such 40S-factor complexes. Thus, three distinct pathways for Met-tRNA(i) delivery to the 40S ribosomal subunit are identified, but which one predominates in vivo remains to be elucidated.

Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3).

Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.

Phosphorylation stoichiometries of human eukaryotic initiation factors.

Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation.

It's a competitive business.

A new in vitro system called Rec-Seq sheds light on how mRNA molecules compete for the machinery that translates their genetic sequence into proteins.

The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A.

Eukaryotic translation initiation involves recruitment of the 43S pre-initiation complex to the 5' end of mRNA by the cap-binding complex eIF4F, forming the 48S translation initiation complex (48S), which then scans along the mRNA until the start codon is recognized. We have previously shown that eIF4F binds near the mRNA exit channel of the 43S, leaving open the question of how mRNA secondary structure is removed as it enters the mRNA channel on the other side of the 40S subunit. Here we report the structure of a human 48S that shows that, in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound at the mRNA entry site, which could unwind RNA secondary structures as they enter the 48S. The structure also reveals conserved interactions between eIF4F and the 43S, probaby explaining how eIF4F can promote mRNA recruitment in all eukaryotes.

Human eukaryotic initiation factor 4G directly binds the 40S ribosomal subunit to promote efficient translation.

Messenger RNA (mRNA) recruitment to the 40S ribosomal subunit is mediated by eukaryotic initiation factor 4F (eIF4F). This complex includes 3 subunits: eIF4E (m 7 G cap binding protein), eIF4A (DEAD box helicase), and eIF4G. Mammalian eIF4G is a scaffold that coordinates the activities of eIF4E and eIF4A and provides a bridge to connect the mRNA and 40S ribosomal subunit through its interaction with eIF3. While the roles of many eIF4G binding domains are relatively clear, the precise function of RNA binding by eIF4G remains to be elucidated. In this work, we used an eIF4G-dependent translation assay to reveal that the RNA binding domain (eIF4G-RBD; amino acids 682-720) stimulates translation. This stimulating activity is observed when eIF4G is independently tethered to an internal region of the mRNA, suggesting that the eIF4G-RBD promotes translation by a mechanism that is independent of the m 7 G cap and mRNA tethering. Using a kinetic helicase assay, we show that the eIF4G-RBD has a minimal effect on eIF4A helicase activity, demonstrating that the eIF4G-RBD is not required to coordinate eIF4F-dependent duplex unwinding. Unexpectedly, native gel electrophoresis and fluorescence polarization assays reveal a previously unidentified direct interaction between eIF4G and the 40S subunit. Using binding assays, our data show that this 40S subunit interaction is separate from the previously characterized interaction between eIF4G and eIF3. Thus, our work reveals how eIF4F can bind to the 40S subunit using eIF3-dependent and eIF3-independent binding domains to promote translation initiation.

Inactivation of eukaryotic initiation factor 5A (eIF5A) by specific acetylation of its hypusine residue by spermidine/spermine acetyltransferase 1 (SSAT1).

eIF5A (eukaryotic translation initiation factor 5A) is the only cellular protein containing hypusine [Nϵ-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine and the hypusine modification is essential for cell proliferation. In the present study, we report selective acetylation of the hypusine and/or deoxyhypusine residue of eIF5A by a key polyamine catabolic enzyme SSAT1 (spermidine/spermine-N1-acetyltransferase 1). This enzyme normally catalyses the N1-acetylation of spermine and spermidine to form acetyl-derivatives, which in turn are degraded to lower polyamines. Although SSAT1 has been reported to exert other effects in cells by its interaction with other cellular proteins, eIF5A is the first target protein specifically acetylated by SSAT1. Hypusine or deoxyhypusine, as the free amino acid, does not act as a substrate for SSAT1, suggesting a macromolecular interaction between eIF5A and SSAT1. Indeed, the binding of eIF5A and SSAT1 was confirmed by pull-down assays. The effect of the acetylation of hypusine on eIF5A activity was assessed by comparison of acetylated with non-acetylated bovine testis eIF5A in the methionyl-puromycin synthesis assay. The loss of eIF5A activity by this SSAT1-mediated acetylation confirms the strict structural requirement for the hypusine side chain and suggests a possible regulation of eIF5A by hypusine acetylation/deacetylation.