[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jan 2014 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: More than one 80S monosome can translate an mRNA molecule at a time producing polysomes. The most widely used method to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes is a high-velocity centrifugation of whole cell extracts in linear sucrose gradients. This polysome profile analysis technique has been routinely used to monitor translational fitness of cells under a variety of physiological conditions, to investigate functions of initiation factors involved in translation, to reveal defects in ribosome biogenesis, to determine roles of 5' UTR structures on mRNA translatability, and more recently for examination of miRNA-mediated translational repression (see an application of this protocol on Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).
No preview · Article · Sep 2013 · Methods in enzymology
[Show abstract][Hide abstract] ABSTRACT: There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the
least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of
robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational
power to computationally explore the mRNA–rRNA complementarity that is statistically significant across the species. Our predictions
reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5′ untranslated regions (UTRs) forming a well-defined
3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5′ UTRs
of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on
18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome
including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context
of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5′ UTRs of mRNAs.
Preview · Article · Jun 2013 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B's domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome's mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.
[Show abstract][Hide abstract] ABSTRACT: Processing bodies (P-bodies) are dynamic cytoplasmic structures involved in mRNA degradation, but the mechanism that governs their formation is poorly understood. In this paper, we address a role of Like-Sm (LSm) proteins in formation of P-bodies and provide evidence that depletion of nuclear LSm8 increases the number of P-bodies, while LSm8 overexpression leads to P-body loss. We show that LSm8 knockdown causes relocalization of LSm4 and LSm6 proteins to the cytoplasm and suggest that LSm8 controls nuclear accumulation of all LSm2-7 proteins. We propose a model in which redistribution of LSm2-7 to the cytoplasm creates new binding sites for other P-body components and nucleates new, microscopically visible structures. The model is supported by prolonged residence of two P-body proteins, DDX6 and Ago2, in P-bodies after LSm8 depletion, which indicates stronger interactions between these proteins and P-bodies. Finally, an increased number of P-bodies has negligible effects on microRNA-mediated translation repression and nonsense mediated decay, further supporting the view that the function of proteins localized in P-bodies is independent of visible P-bodies.
Full-text · Article · Aug 2012 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Jun 2012 · Current Protein and Peptide Science
[Show abstract][Hide abstract] ABSTRACT: 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 sui1G107R 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.
Full-text · Article · Nov 2011 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: In eukaryotes, the 40 S ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely
on the AUG start codon. Structural arrangement of the 18 S rRNA determines the overall shape of the 40 S subunit. Here, we
present genetic evaluation of yeast 18 S rRNA function using 10 point mutations altering the polysome profile. All the mutants
reduce the abundance of the mutant 40 S, making it limiting for translation initiation. Two of the isolated mutations, G875A, altering the core of the platform domain that binds eIF1 and eIF2, and A1193U, changing the h31 loop located below the P-site tRNAiMet, show phenotypes indicating defective regulation of AUG selection. Evidence is provided that these mutations reduce the interaction
with the components of the preinitiation complex, thereby inhibiting its function at different steps. These results indicate
that the 18 S rRNA mutations impair the integrity of scanning-competent preinitiation complex, thereby altering the 40 S subunit
response to stringent AUG selection. Interestingly, nine of the mutations alter the body/platform domains of 18 S rRNA, potentially
affecting the bridges to the 60 S subunit, but they do not change the level of 18 S rRNA intermediates. Based on these results,
we also discuss the mechanism of the selective degradation of the mutant 40 S subunits.
Preview · Article · Oct 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Oct 2010 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: The C-terminal domain (CTD) of the a/Tif32 subunit of budding yeast eukaryotic translation initiation factor 3 (eIF3) interacts
with eIF3 subunits j/Hcr1 and b/Prt1 and can bind helices 16 to 18 of 18S rRNA, suggesting proximity to the mRNA entry channel
of the 40S subunit. We have identified substitutions in the conserved Lys-Glu-Arg-Arg (KERR) motif and in residues of the
nearby box6 element of the a/Tif32 CTD that impair mRNA recruitment by 43S preinitiation complexes (PICs) and confer phenotypes
indicating defects in scanning and start codon recognition. The normally dispensable CTD of j/Hcr1 is required for its binding
to a/Tif32 and to mitigate the growth defects of these a/Tif32 mutants, indicating physical and functional interactions between
these two domains. The a/Tif32 CTD and the j/Hcr1 N-terminal domain (NTD) also interact with the RNA recognition motif (RRM)
in b/Prt1, and mutations in both subunits that disrupt their interactions with the RRM increase leaky scanning of an AUG codon.
These results, and our demonstration that the extreme CTD of a/Tif32 binds to Rps2 and Rps3, lead us to propose that the a/Tif32
CTD directly stabilizes 43S subunit-mRNA interaction and that the b/Prt1-RRM-j/Hcr1-a/Tif32-CTD module binds near the mRNA
entry channel and regulates the transition between scanning-conducive and initiation-competent conformations of the PIC.
[Show abstract][Hide abstract] ABSTRACT: Despite recent progress in our understanding of the numerous functions of individual subunits of eukaryotic translation initiation factor (eIF) 3, little is known on the molecular level. Using NMR spectroscopy, we determined the first solution structure of an interaction between eIF3 subunits. We revealed that a conserved tryptophan residue in the human eIF3j N-terminal acidic motif (NTA) is held in the helix alpha1 and loop 5 hydrophobic pocket of the human eIF3b RNA recognition motif (RRM). Mutating the corresponding "pocket" residues in its yeast orthologue reduces cellular growth rate, eliminates eIF3j/HCR1 association with eIF3b/PRT1 in vitro and in vivo, affects 40S occupancy of eIF3, and produces a leaky scanning defect indicative of a deregulation of the AUG selection process. Unexpectedly, we found that the N-terminal half of eIF3j/HCR1 containing the NTA is indispensable and sufficient for wild-type growth of yeast cells. Furthermore, we demonstrate that deletion of either j/HCR1 or its N-terminal half only, or mutation of the key tryptophan residues results in the severe leaky scanning phenotype partially suppressible by overexpressed eIF1A, which is thought to stabilize properly formed preinitiation complexes at the correct start codon. These findings indicate that eIF3j/HCR1 remains associated with the scanning preinitiation complexes and does not dissociate from the small ribosomal subunit upon mRNA recruitment, as previously believed. Finally, we provide further support for earlier mapping of the ribosomal binding site for human eIF3j by identifying specific interactions of eIF3j/HCR1 with small ribosomal proteins RPS2 and RPS23 located in the vicinity of the mRNA entry channel. Taken together, we propose that eIF3j/HCR1 closely cooperates with the eIF3b/PRT1 RRM and eIF1A on the ribosome to ensure proper formation of the scanning-arrested conformation required for stringent AUG recognition.
Full-text · Article · Mar 2010 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Environmental stresses inducing translation arrest are accompanied by the deposition of translational components into stress granules (SGs) serving as mRNA triage sites. It has recently been reported that, in Saccharomyces cerevisiae, formation of SGs occurs as a result of a prolonged glucose starvation. However, these SGs did not contain eIF3, one of hallmarks of mammalian SGs. We have analyzed the effect of robust heat shock on distribution of eIF3a/Tif32p/Rpg1p and showed that it results in the formation of eIF3a accumulations containing other eIF3 subunits, known yeast SG components and small but not large ribosomal subunits and eIF2alpha/Sui2p. Interestingly, under these conditions, Dcp2p and Dhh1p P-body markers also colocalized with eIF3a. Microscopic analyses of the edc3Deltalsm4DeltaC mutant demonstrated that different scaffolding proteins are required to induce SGs upon robust heat shock as opposed to glucose deprivation. Even though eIF2alpha became phosphorylated under these stress conditions, the decrease in polysomes and formation of SGs occurred independently of phosphorylation of eIF2alpha. We conclude that under specific stress conditions, such as robust heat shock, yeast SGs do contain eIF3 and 40S ribosomes and utilize alternative routes for their assembly.
[Show abstract][Hide abstract] ABSTRACT: Yeast initiation factor eIF3 (eukaryotic initiation factor 3) has been implicated in multiple steps of translation initiation. Previously, we showed that the N-terminal domain (NTD) of eIF3a interacts with the small ribosomal protein RPS0A located near the mRNA exit channel, where eIF3 is proposed to reside. Here, we demonstrate that a partial deletion of the RPS0A-binding domain of eIF3a impairs translation initiation and reduces binding of eIF3 and associated eIFs to native preinitiation complexes in vivo. Strikingly, it also severely blocks the induction of GCN4 translation that occurs via reinitiation. Detailed examination unveiled a novel reinitiation defect resulting from an inability of 40S ribosomes to resume scanning after terminating at the first upstream ORF (uORF1). Genetic analysis reveals a functional interaction between the eIF3a-NTD and sequences 5' of uORF1 that is critically required to enhance reinitiation. We further demonstrate that these stimulatory sequences must be positioned precisely relative to the uORF1 stop codon and that reinitiation efficiency after uORF1 declines with its increasing length. Together, our results suggest that eIF3 is retained on ribosomes throughout uORF1 translation and, upon termination, interacts with its 5' enhancer at the mRNA exit channel to stabilize mRNA association with post-termination 40S subunits and enable resumption of scanning for reinitiation downstream.
Full-text · Article · Oct 2008 · Genes & Development
[Show abstract][Hide abstract] ABSTRACT: Translation initiation starts with the formation of the 43S preinitiation complex (PIC) consisting of several soluble factors, including the ternary complex (TC; elF2-GTP-Met-tRNA(i)(Met)), which associate with the small ribosomal subunit. In the next step, mRNA is recruited to form the 48S PIC and the entire machinery starts scanning the 5' untranslated region of the mRNA until the AUG start codon is encountered. The most widely used method to separate 40S and 60S ribosomal subunits from soluble factors, monosomes and polysomes, is sucrose density centrifugation (SDC). Since PICs are intrinsically unstable complexes that cannot withstand the forces imposed by SDC, a stabilization agent must be employed to detect the association of factors with the 40S subunit after SDC. This was initially achieved by adding heparin (a highly sulfated glycosaminoglycan) directly to the breaking buffer of cells treated with cycloheximide (a translation elongation inhibitor). However, the mechanism of stabilization is not understood and, moreover, there are indications that the use of heparin may lead to artifactual factor associations that do not reflect the factor occupancy of the 43S/48S PICs in the cell at the time of lysis. Therefore, we developed an alternative method for PIC stabilization using formaldehyde (HCHO) to cross-link factors associated with 40S ribosomal subunits in vivo before the disruption of the yeast cells. Results obtained using HCHO stabilization strongly indicate that the factors detected on the 43S/48S PIC after SDC approximate a real-time in vivo "snapshot" of the 43S/48S PIC composition. In this chapter, we will present the protocol for HCHO cross-linking in detail and demonstrate the difference between heparin and HCHO stabilization procedures. In addition, different conditions for displaying the polysome profile or PIC analysis by SDC, used to address different questions, will be outlined.
Full-text · Article · Feb 2007 · Methods in Enzymology
[Show abstract][Hide abstract] ABSTRACT: Protein complexes play a critical role in virtually all cellular processes that have been studied to date. Comprehensive knowledge of the architecture of a protein complex of interest is, therefore, an important prerequisite for understanding its role in the context of a particular pathway in which it participates. One of the possible approaches that has proven very useful in characterizing a protein complex is outlined in this chapter using the example of the eukaryotic initiation factor 3 (eIF3) and some of its binding partners. eIF3 is one of the major players in the translation initiation pathway because it orchestrates several crucial steps that ultimately conclude with formation of the 80S ribosome where the anticodon of methionyl-tRNA(i)(Met) base-pairs with the AUG start codon of the mRNA in the ribosomal P-site. We previously demonstrated that, in the budding yeast Saccharomyces cerevisiae, eIF3 closely cooperates with several other eIFs to stimulate recruitment of methionyl-tRNA(i)(Met) and mRNA to the 40S ribosome and that it forms, together with eIFs 1, 2, and 5, an important intermediate in translation initiation called the multifactor complex (MFC). Here, we summarize the fundamental procedure that allowed in-depth characterization of the MFC composition and identification of protein-protein interactions among its constituents. Primarily, we describe in detail in vivo purification techniques that, in combination with systematic deletion analysis, produced a 3D subunit interaction model for the MFC. Site-directed clustered-10-alanine-mutagenesis (CAM) employed to investigate the physiological significance of individual interactions is also presented. The general character of the entire procedure makes it usable for first-order structural characterization of virtually any soluble protein complex in yeast.
Full-text · Article · Feb 2007 · Methods in Enzymology
[Show abstract][Hide abstract] ABSTRACT: We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1
(prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation in vivo and reduces 40S-binding of eIF3 to native preinitiation complexes.
Several findings indicate that the rnp1 lesion decreases recruitment of eIF3 to the 40S subunit by HCR1: (i) rnp1 strongly impairs the association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (ii) rnp1 impairs the 40S binding of eIF3 more so than the 40S binding of HCR1; (iii) overexpressing HCR1-R215I decreases the Ts− phenotype and increases 40S-bound eIF3 in rnp1 cells; (iv) the rnp1 Ts− phenotype is exacerbated by tif32-Δ6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1Δ impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also reduces the levels of 40S-bound eIF5 and eIF1 and increases leaky scanning at the GCN4 uORF1. Thus, the PRT1 RNP1 motif coordinates the functions of HCR1 and TIF32 in 40S binding of eIF3 and is needed for optimal
preinitiation complex assembly and AUG recognition in vivo.
Full-text · Article · May 2006 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and
eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between
the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects
of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents
of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding
by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was
reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit
joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free
40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and
eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and
that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.
Full-text · Article · Mar 2006 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic cells respond to starvation by decreasing the rate of general protein synthesis while inducing translation of specific mRNAs encoding transcription factors GCN4 (yeast) or ATF4 (humans). Both responses are elicited by phosphorylation of translation initiation factor 2 (eIF2) and the attendant inhibition of its nucleotide exchange factor eIF2B-decreasing the binding to 40S ribosomes of methionyl initiator tRNA in the ternary complex (TC) with eIF2 and GTP. The reduction in TC levels enables scanning ribosomes to bypass the start codons of upstream open reading frames in the GCN4 mRNA leader and initiate translation at the authentic GCN4 start codon. We exploited the fact that GCN4 translation is a sensitive reporter of defects in TC recruitment to identify the catalytic and regulatory subunits of eIF2B. More recently, we implicated the C-terminal domain of eIF1A in 40S-binding of TC in vivo. Interestingly, we found that TC resides in a multifactor complex (MFC) with eIF3, eIF1, and the GTPase-activating protein for eIF2, known as eIF5. Our biochemical and genetic analyses indicate that physical interactions between MFC components enhance TC binding to 40S subunits and are required for wild-type translational control of GCN4. MFC integrity and eIF3 function also contribute to post-assembly steps in the initiation pathway that impact GCN4 expression. Thus, apart from its critical role in the starvation response, GCN4 regulation is a valuable tool for dissecting the contributions of multiple translation factors in the eukaryotic initiation pathway.
No preview · Article · Jan 2005 · Annals of the New York Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The N-terminal domain (NTD) of NIP1/eIF3c interacts directly with eIF1 and eIF5 and indirectly through eIF5 with the eIF2-GTP-Met-tRNA(i)(Met) ternary complex (TC) to form the multifactor complex (MFC). We investigated the physiological importance of these interactions by mutating 16 segments spanning the NIP1-NTD. Mutations in multiple segments reduced the binding of eIF1 or eIF5 to the NIP1-NTD. Mutating a C-terminal segment of the NIP1-NTD increased utilization of UUG start codons (Sui(-) phenotype) and was lethal in cells expressing eIF5-G31R that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons. Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui(-) phenotype conferred by eIF5-G31R. Mutations in two N-terminal segments of the NIP1-NTD suppressed the Sui(-) phenotypes produced by the eIF1-D83G and eIF5-G31R mutations. From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons. Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes. Thus, the NIP1-NTD is required for efficient assembly of preinitiation complexes and also regulates the selection of AUG start codons in vivo.
Full-text · Article · Dec 2004 · Molecular and Cellular Biology