eIF3 is a large multiprotein complex serving as an essential scaffold promoting binding of other eIFs to the 40S subunit, where it coordinates their actions during translation initiation. Perhaps due to a high degree of flexibility of multiple eIF3 subunits, a high-resolution structure of free eIF3 from any organism has never been solved. Employing genetics and biochemistry, we previously built a 2D interaction map of all five yeast eIF3 subunits. Here we further improved the previously reported in vitro reconstitution protocol of yeast eIF3, which we cross-linked and trypsin-digested to determine its overall shape in 3D by advanced mass-spectrometry. The obtained cross-links support our 2D subunit interaction map and reveal that eIF3 is tightly packed with its WD40 and RRM domains exposed. This contrasts with reported cryo-EM structures depicting eIF3 as a molecular embracer of the 40S subunit. Since the binding of eIF1 and eIF5 further fortified the compact architecture of eIF3, we suggest that its initial contact with the 40S solvent-exposed side makes eIF3 to open up and wrap around the 40S head with its extended arms. In addition, we mapped the position of eIF5 to the region below the P- and E-sites of the 40S subunit.

• MeSH
• elektronová kryomikroskopie MeSH
• eukaryotický iniciační faktor 1 chemie   genetika   metabolismus   MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• eukaryotický iniciační faktor 5 chemie   genetika   metabolismus   MeSH
• iniciace translace peptidového řetězce * MeSH
• malé podjednotky ribozomu eukaryotické genetika   metabolismus   MeSH
• molekulární modely MeSH
• proteinové domény MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   ultrastruktura   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eukaryotický iniciační faktor 1 MeSH
• eukaryotický iniciační faktor 3 MeSH
• eukaryotický iniciační faktor 5 MeSH
• Saccharomyces cerevisiae - proteiny MeSH

Protein synthesis is mediated via numerous molecules including the ribosome, mRNA, tRNAs, as well as translation initiation, elongation and release factors. Some of these factors play several roles throughout the entire process to ensure proper assembly of the preinitiation complex on the right mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most intriguing of these multitasking factors is the eukaryotic initiation factor eIF3. Recent evidence strongly suggests that this factor, which coordinates the progress of most of the initiation steps, does not come off the initiation complex upon subunit joining, but instead it remains bound to 80S ribosomes and gradually falls off during the first few elongation cycles to: (1) promote resumption of scanning on the same mRNA molecule for reinitiation downstream-in case of translation of upstream ORFs short enough to preserve eIF3 bound; or (2) come back during termination on long ORFs to fine tune its fidelity or, if signaled, promote programmed stop codon readthrough. Here, we unite recent structural views of the eIF3-40S complex and discus all known eIF3 roles to provide a broad picture of the eIF3's impact on translational control in eukaryotic cells.

• MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• konformace proteinů * MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární modely MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• proteosyntéza * MeSH
• ribozomy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• messenger RNA MeSH
• podjednotky proteinů MeSH
• Saccharomyces cerevisiae - proteiny MeSH

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.

• Klíčová slova
• S. cerevisiae, biochemistry, biophysics, eIF3, initiation, mRNA recruitment, ribosome, structural biology, translation, yeast,
• MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• guanosintrifosfát metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• mutační analýza DNA MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• podjednotky proteinů genetika   metabolismus   MeSH
• proteosyntéza MeSH
• ribozomy metabolismus   MeSH
• RNA transferová Met metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• guanosintrifosfát MeSH
• messenger RNA MeSH
• mutantní proteiny MeSH
• podjednotky proteinů MeSH
• RNA transferová Met MeSH

Transfer of genetic information from genes into proteins is mediated by messenger RNA (mRNA) that must be first recruited to ribosomal pre-initiation complexes (PICs) by a mechanism that is still poorly understood. Recent studies showed that besides eIF4F and poly(A)-binding protein, eIF3 also plays a critical role in this process, yet the molecular mechanism of its action is unknown. We showed previously that the PCI domain of the eIF3c/NIP1 subunit of yeast eIF3 is involved in RNA binding. To assess the role of the second PCI domain of eIF3 present in eIF3a/TIF32, we performed its mutational analysis and identified a 10-Ala-substitution (Box37) that severely reduces amounts of model mRNA in the 43-48S PICs in vivo as the major, if not the only, detectable defect. Crystal structure analysis of the a/TIF32-PCI domain at 2.65-Å resolution showed that it is required for integrity of the eIF3 core and, similarly to the c/NIP1-PCI, is capable of RNA binding. The putative RNA-binding surface defined by positively charged areas contains two Box37 residues, R363 and K364. Their substitutions with alanines severely impair the mRNA recruitment step in vivo suggesting that a/TIF32-PCI represents one of the key domains ensuring stable and efficient mRNA delivery to the PICs.

• MeSH
• alanin genetika   MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• fenotyp MeSH
• iniciace translace peptidového řetězce * MeSH
• malé podjednotky ribozomu eukaryotické metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• molekulární modely MeSH
• mutace MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• terciární struktura proteinů MeSH
• transkripční faktory bZIP genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• eukaryotický iniciační faktor 3 MeSH
• GCN4 protein, S cerevisiae MeSH Prohlížeč
• messenger RNA MeSH
• NIP1 protein, S cerevisiae MeSH Prohlížeč
• RPG1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• transkripční faktory bZIP MeSH

In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5'-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1-45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60-137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site.

• MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• iniciace translace peptidového řetězce fyziologie   MeSH
• kodon iniciační genetika   metabolismus   MeSH
• malé podjednotky ribozomu eukaryotické genetika   metabolismus   MeSH
• multiproteinové komplexy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• kodon iniciační MeSH
• multiproteinové komplexy MeSH
• NIP1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH

The ribosome translates information encoded by mRNAs into proteins in all living cells. In eukaryotes, its small subunit together with a number of eukaryotic initiation factors (eIFs) is responsible for locating the mRNA's translational start to properly decode the genetic message that it carries. This multistep process requires timely and spatially coordinated placement of eIFs on the ribosomal surface. In our long-standing pursuit to map the 40S-binding site of one of the functionally most complex eIFs, yeast multisubunit eIF3, we identified several interactions that placed its major body to the head, beak and shoulder regions of the solvent-exposed side of the 40S subunit. Among them is the interaction between the N-terminal domain (NTD) of the a/TIF32 subunit of eIF3 and the small ribosomal protein RPS0A, residing near the mRNA exit channel. Previously, we demonstrated that the N-terminal truncation of 200 residues in tif32-Δ8 significantly reduced association of eIF3 and other eIFs with 40S ribosomes in vivo and severely impaired translation reinitiation that eIF3 ensures. Here we show that not the first but the next 200 residues of a/TIF32 specifically interact with RPS0A via its extreme C-terminal tail (CTT). Detailed analysis of the RPS0A conditional depletion mutant revealed a marked drop in the polysome to monosome ratio suggesting that the initiation rates of cells grown under non-permissive conditions were significantly impaired. Indeed, amounts of eIF3 and other eIFs associated with 40S subunits in the pre-initiation complexes in the RPS0A-depleted cells were found reduced; consistently, to the similar extent as in the tif32-Δ8 cells. Similar but less pronounced effects were also observed with the viable CTT-less mutant of RPS0A. Together we conclude that the interaction between the flexible RPS0A-CTT and the residues 200-400 of the a/TIF32-NTD significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo.

• MeSH
• eukaryotický iniciační faktor 3 metabolismus   MeSH
• genový knockout MeSH
• iniciace translace peptidového řetězce * MeSH
• interakční proteinové domény a motivy MeSH
• malé podjednotky ribozomu eukaryotické metabolismus   MeSH
• podjednotky proteinů metabolismus   MeSH
• ribozomální proteiny genetika   metabolismus   fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   fyziologie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• techniky dvojhybridového systému MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• podjednotky proteinů MeSH
• ribozomální proteiny MeSH
• RPG1 protein, S cerevisiae MeSH Prohlížeč
• RPS0A protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH

Protein synthesis is a fundamental biological mechanism bringing the DNA-encoded genetic information into life by its translation into molecular effectors - proteins. The initiation phase of translation is one of the key points of gene regulation in eukaryotes, playing a role in processes from neuronal function to development. Indeed, the importance of the study of protein synthesis is increasing with the growing list of genetic diseases caused by mutations that affect mRNA translation. To grasp how this regulation is achieved or altered in the latter case, we must first understand the molecular details of all underlying processes of the translational cycle with the main focus put on its initiation. In this review I discuss recent advances in our comprehension of the molecular basis of particular initiation reactions set into the context of how and where individual eIFs bind to the small ribosomal subunit in the pre-initiation complex. I also summarize our current knowledge on how eukaryotic initiation factor eIF3 controls gene expression in the gene-specific manner via reinitiation.

• MeSH
• Eukaryota metabolismus   MeSH
• eukaryotické iniciační faktory metabolismus   MeSH
• iniciace translace peptidového řetězce genetika   MeSH
• lidé MeSH
• molekulární modely MeSH
• ribozomy metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• eukaryotické iniciační faktory MeSH

Several subunits of the multifunctional eukaryotic translation initiation factor 3 (eIF3) contain well-defined domains. Among them is the conserved bipartite PCI domain, typically serving as the principal scaffold for multisubunit 26S proteasome lid, CSN and eIF3 complexes, which constitutes most of the C-terminal region of the c/NIP1 subunit. Interestingly, the c/NIP1-PCI domain is exceptional in that its deletion, despite being lethal, does not affect eIF3 integrity. Here, we show that a short C-terminal truncation and two clustered mutations directly disturbing the PCI domain produce lethal or slow growth phenotypes and significantly reduce amounts of 40S-bound eIF3 and eIF5 in vivo. The extreme C-terminus directly interacts with blades 1-3 of the small ribosomal protein RACK1/ASC1, which is a part of the 40S head, and, consistently, deletion of the ASC1 coding region likewise affects eIF3 association with ribosomes. The PCI domain per se shows strong but unspecific binding to RNA, for the first time implicating this typical protein-protein binding domain in mediating protein-RNA interactions also. Importantly, as our clustered mutations severely reduce RNA binding, we conclude that the c/NIP1 C-terminal region forms an important intermolecular bridge between eIF3 and the 40S head region by contacting RACK1/ASC1 and most probably 18S rRNA.

• MeSH
• adaptorové proteiny signální transdukční chemie   genetika   metabolismus   MeSH
• delece genu MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• iniciace translace peptidového řetězce * MeSH
• interakční proteinové domény a motivy MeSH
• malé podjednotky ribozomu eukaryotické chemie   metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• proteiny vázající GTP chemie   genetika   metabolismus   MeSH
• RNA ribozomální 18S metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny biosyntéza   chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• transkripční faktory bZIP biosyntéza   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• ASC1 protein, S cerevisiae MeSH Prohlížeč
• eukaryotický iniciační faktor 3 MeSH
• GCN4 protein, S cerevisiae MeSH Prohlížeč
• NIP1 protein, S cerevisiae MeSH Prohlížeč
• podjednotky proteinů MeSH
• proteiny vázající GTP MeSH
• RNA ribozomální 18S MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• transkripční faktory bZIP MeSH

Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1(G107R) but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

• MeSH
• eukaryotický iniciační faktor 1 genetika   MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• fenotyp MeSH
• genová dávka MeSH
• iniciace translace peptidového řetězce * MeSH
• kodon iniciační MeSH
• krystalografie rentgenová MeSH
• kvasinky genetika   růst a vývoj   MeSH
• malé podjednotky ribozomu eukaryotické metabolismus   MeSH
• molekulární modely MeSH
• mutace MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eukaryotic peptide initiation factor-1A MeSH Prohlížeč
• eukaryotický iniciační faktor 1 MeSH
• eukaryotický iniciační faktor 3 MeSH
• kodon iniciační MeSH
• Prt1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• TIF34 protein, S cerevisiae MeSH Prohlížeč

Recent reports have begun unraveling the details of various roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits in translation initiation. Here we describe functional characterization of two essential Saccharomyces cerevisiae eIF3 subunits, g/Tif35 and i/Tif34, previously suggested to be dispensable for formation of the 48S preinitiation complexes (PICs) in vitro. A triple-Ala substitution of conserved residues in the RRM of g/Tif35 (g/tif35-KLF) or a single-point mutation in the WD40 repeat 6 of i/Tif34 (i/tif34-Q258R) produces severe growth defects and decreases the rate of translation initiation in vivo without affecting the integrity of eIF3 and formation of the 43S PICs in vivo. Both mutations also diminish induction of GCN4 expression, which occurs upon starvation via reinitiation. Whereas g/tif35-KLF impedes resumption of scanning for downstream reinitiation by 40S ribosomes terminating at upstream open reading frame 1 (uORF1) in the GCN4 mRNA leader, i/tif34-Q258R prevents full GCN4 derepression by impairing the rate of scanning of posttermination 40S ribosomes moving downstream from uORF1. In addition, g/tif35-KLF reduces processivity of scanning through stable secondary structures, and g/Tif35 specifically interacts with Rps3 and Rps20 located near the ribosomal mRNA entry channel. Together these results implicate g/Tif35 and i/Tif34 in stimulation of linear scanning and, specifically in the case of g/Tif35, also in proper regulation of the GCN4 reinitiation mechanism.

• MeSH
• aminokyselinové motivy genetika   MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• fungální RNA metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• proteosyntéza * MeSH
• ribozomální proteiny chemie   genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• substituce aminokyselin MeSH
• terciární struktura proteinů MeSH
• transkripční faktory bZIP genetika   metabolismus   MeSH
• vazba proteinů MeSH
• western blotting MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• fungální RNA MeSH
• GCN4 protein, S cerevisiae MeSH Prohlížeč
• messenger RNA MeSH
• podjednotky proteinů MeSH
• ribozomální proteiny MeSH
• RPS20 protein, S cerevisiae MeSH Prohlížeč
• RPS3 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• transkripční faktory bZIP MeSH