Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP.
ABSTRACT: The 40S subunit in 48S complexes formed at the initiation codon of mRNA is bound to eukaryotic initiation factor (eIF) 3, eIF1, eIF1A, and an eIF2/GTP/Met-tRNAi(Met) ternary complex and can therefore not join a 60S subunit directly to form an 80S ribosome. We report that eIF5-induced hydrolysis of eIF2-bound GTP in 48S complexes led to release of eIF2-GDP but not eIF3 or eIF1. eIF5B did not influence factor release in the absence of 60S subunits. Therefore eIF3 and eIF1 dissociate from 40S subunits during, rather than before, the eIF5B-mediated subunit joining event. In the absence of eIF1, eIF5-stimulated hydrolysis of eIF2-bound GTP occurred at the same rate in 43S pre-initiation and 48S initiation complexes. GTP hydrolysis in 43S complexes assembled with eIF1 was much slower than in 43S or 48S complexes assembled without eIF1. Establishment of codon-anticodon base-pairing in 48S complexes relieved eIF1's inhibition. Thus, in addition to its role in initiation codon selection during 48S complex formation, eIF1 also participates in maintaining the fidelity of the initiation process at a later stage, hydrolysis of eIF2-bound GTP, by inhibiting premature GTP hydrolysis and by linking establishment of codon-anticodon base-pairing with GTP hydrolysis.
Project description:48S initiation complex (48S IC) formation is the first stage in the eukaryotic translation process. According to the canonical mechanism, 40S ribosomal subunit binds to the 5'-end of messenger RNA (mRNA) and scans its 5'-untranslated region (5'-UTR) to the initiation codon where it forms the 48S IC. Entire process is mediated by initiation factors. Here we show that eIF5 and eIF5B together stimulate 48S IC formation influencing initiation codon selection during ribosomal scanning. Initiation on non-optimal start codons--following structured 5'-UTRs, in bad AUG context, within few nucleotides from 5'-end of mRNA and CUG start codon--is the most affected. eIF5-induced hydrolysis of eIF2-bound GTP is essential for stimulation. GTP hydrolysis increases the probability that scanning ribosomal complexes will recognize and arrest scanning at a non-optimal initiation codon. Such 48S ICs are less stable owing to dissociation of eIF2*GDP from initiator tRNA, and eIF5B is then required to stabilize the initiator tRNA in the P site of 40S subunit. Alternative model that eIF5 and eIF5B cause 43S pre-initiation complex rearrangement favoring more efficient initiation codon recognition during ribosomal scanning is equally possible. Mutational analysis of eIF1A and eIF5B revealed distinct functions of eIF5B in 48S IC formation and subunit joining.
Project description:During eukaryotic translation initiation, 43S ribosomal complex scans mRNA leader unless an AUG codon in an appropriate context is found. Establishing the stable codon-anticodon base-pairing traps the ribosome on the initiator codon and triggers structural rearrangements, which lead to Pi release from the eIF2-bound GTP. It is generally accepted that AUG recognition by the scanning 43S complex sets the final point in the process of start codon selection, while latter stages do not contribute to this process. Here we use translation reconstitution approach and kinetic toe-printing assay to show that after the 48S complex is formed on an AUG codon, in case GTP hydrolysis is impaired, the ribosomal subunit is capable to resume scanning and slides downstream to the next AUG. In contrast to leaky scanning, this sliding is not limited to AUGs in poor nucleotide contexts and occurs after a relatively long pause at the recognized AUG. Thus, recognition of an AUG per se does not inevitably lead to this codon being selected for initiation of protein synthesis. Instead, it is eIF5-induced GTP hydrolysis and Pi release that irreversibly trap the 48S complex, and this complex is further stabilized by eIF5B and 60S joining.
Project description:Eukaryotic protein synthesis begins with assembly of 48S initiation complexes at the initiation codon of mRNA, which requires at least seven initiation factors (eIFs). First, 43S preinitiation complexes comprising 40S ribosomal subunits, eIFs 3, 2, 1, and 1A, and tRNA(Met)(i) attach to the 5'-proximal region of mRNA and then scan along the 5' untranslated region (5'UTR) to the initiation codon. Attachment of 43S complexes is mediated by three other eIFs, 4F, 4A, and 4B, which cooperatively unwind the cap-proximal region of mRNA and later also assist 43S complexes during scanning. We now report that these seven eIFs are not sufficient for efficient 48S complex formation on mRNAs with highly structured 5'UTRs, and that this process requires the DExH-box protein DHX29. DHX29 binds 40S subunits and hydrolyzes ATP, GTP, UTP, and CTP. NTP hydrolysis by DHX29 is strongly stimulated by 43S complexes and is required for DHX29's activity in promoting 48S complex formation.
Project description:During eukaryotic translation initiation, the 43S preinitiation complex (43S PIC), consisting of the 40S ribosomal subunit, eukaryotic initiation factors (eIFs) and initiator tRNA scans mRNA to find an appropriate start codon. Key roles in the accuracy of initiation codon selection belong to eIF1 and eIF1A, whereas the mammalian-specific DHX29 helicase substantially contributes to ribosomal scanning of structured mRNAs. Here, we show that DHX29 stimulates the recognition of the AUG codon but not the near-cognate CUG codon regardless of its nucleotide context during ribosomal scanning. The stimulatory effect depends on the contact between DHX29 and eIF1A. The unique DHX29 N-terminal domain binds to the ribosomal site near the mRNA entrance, where it contacts the eIF1A OB domain. UV crosslinking assays revealed that DHX29 may rearrange eIF1A and eIF2? in key nucleotide context positions of ribosomal complexes. Interestingly, DHX29 impedes the 48S initiation complex formation in the absence of eIF1A perhaps due to forming a physical barrier that prevents the 43S PIC from loading onto mRNA. Mutational analysis allowed us to split the mRNA unwinding and codon selection activities of DHX29. Thus, DHX29 is another example of an initiation factor contributing to start codon selection.
Project description:Halastavi árva virus (HalV) has a positive-sense RNA genome, with an 827 nt-long 5' UTR and an intergenic region separating two open reading frames. Whereas the encoded proteins are most homologous to Dicistrovirus polyproteins, its 5' UTR is distinct. Here, we report that the HalV 5' UTR comprises small stem-loop domains separated by long single-stranded areas and a large A-rich unstructured region surrounding the initiation codon AUG828, and possesses cross-kingdom internal ribosome entry site (IRES) activity. In contrast to most viral IRESs, it does not depend on structural integrity and specific interaction of a structured element with a translational component, and is instead determined by the unstructured region flanking AUG828. eIF2, eIF3, eIF1 and eIF1A promote efficient 48S initiation complex formation at AUG828, which is reduced ?5-fold on omission of eIF1 and eIF1A. Initiation involves direct attachment of 43S preinitiation complexes within a short window at or immediately downstream of AUG828. 40S and eIF3 are sufficient for initial binding. After attachment, 43S complexes undergo retrograde scanning, strongly dependent on eIF1 and eIF1A. eIF4A/eIF4G stimulated initiation only at low temperatures or on mutants, in which areas surrounding AUG828 had been replaced by heterologous sequences. However, they strongly promoted initiation at AUG872, yielding a proline-rich oligopeptide.
Project description: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.
Project description:Initiation codon selection in eukaryotes involves base-by-base inspection of the 5'-untranslated region of mRNA by scanning ribosomal 43S preinitiation complexes. We employed in vitro reconstitution to investigate factor requirements for this process and report that in the absence of eIF1 and DHX29, eIFs 4A, 4B and 4G promote efficient bypassing of stable stems by scanning 43S complexes and formation of 48S initiation complexes on AUG codons immediately upstream and downstream of such stems, without their unwinding. However, intact stems are not threaded through the entire mRNA Exit channel of the 40S subunit, resulting in incorrect positioning of mRNA upstream of the ribosomal P site in 48S complexes formed on AUG codons following intact stems, which renders them susceptible to dissociation by eIF1. In 48S complexes formed on AUG codons preceding intact stems, the stems are accommodated in the A site. Such aberrant complexes are destabilized by DHX29, which also ensures that mRNA enters the mRNA-binding cleft in a single-stranded form and therefore undergoes base-by-base inspection during scanning.
Project description:The Simian picornavirus type 9 (SPV9) 5'-untranslated region (5' UTR) has been predicted to contain an internal ribosomal entry site (IRES) with structural elements that resemble domains of hepacivirus/pestivirus (HP) IRESs. In vitro reconstitution of initiation confirmed that this 5' UTR contains an IRES and revealed that it has both functional similarities and differences compared to HP IRESs. Like HP IRESs, the SPV9 IRES bound directly to 40S subunits and eukaryotic initiation factor (eIF) 3, depended on the conserved domain IIId for ribosomal binding and consequently for function, and additionally required eIF2/initiator tRNA to yield 48S complexes that formed elongation-competent 80S ribosomes in the presence of eIF5, eIF5B, and 60S subunits. Toeprinting analysis revealed that eIF1A stabilized 48S complexes, whereas eIF1 induced conformational changes in the 40S subunit, likely corresponding to partial opening of the entry latch of the mRNA-binding channel, that were exacerbated by eIF3 and suppressed by eIF1A. The SPV9 IRES differed from HP IRESs in that its function was enhanced by eIF4A/eIF4F when the IRES was adjacent to the wild-type coding sequence, but was less affected by these factors or by a dominant negative eIF4A mutant when potentially less structured coding sequences were present. Exceptionally, this IRES promoted binding of initiator tRNA to the initiation codon in the P site of 40S subunits independently of eIF2. Although these 40S/IRES/tRNA complexes could not form active 80S ribosomes, this constitutes a second difference between the SPV9 and HP IRESs. eIF1 destabilized the eIF2-independent ribosomal binding of initiator tRNA.
Project description:The eukaryotic 43S pre-initiation complex bearing tRNAi(Met) scans the mRNA leader for an AUG start codon in favorable context. Structural analyses revealed that the ?-hairpin of 40S protein Rps5/uS7 protrudes into the 40S mRNA exit-channel, contacting the eIF2?GTP?Met-tRNAi ternary complex (TC) and mRNA context nucleotides; but its importance in AUG selection was unknown. We identified substitutions in ?-strand-1 and C-terminal residues of yeast Rps5 that reduced bulk initiation, conferred 'leaky-scanning' of AUGs; and lowered initiation fidelity by exacerbating the effect of poor context of the eIF1 AUG codon to reduce eIF1 abundance. Consistently, the ?-strand-1 substitution greatly destabilized the 'PIN' conformation of TC binding to reconstituted 43S·mRNA complexes in vitro. Other substitutions in ?-hairpin loop residues increased initiation fidelity and destabilized PIN at UUG, but not AUG start codons. We conclude that the Rps5 ?-hairpin is as crucial as soluble initiation factors for efficient and accurate start codon recognition.
Project description:Eukaryotic translation initiation is a multistep process requiring a number of eukaryotic translation initiation factors (eIFs). Two GTPases play key roles in the process. eIF2 brings the initiator Met-tRNAi to the preinitiation complex (PIC). Upon start codon selection and GTP hydrolysis promoted by the GTPase-activating protein (GAP) eIF5, eIF2-GDP is displaced from Met-tRNAi by eIF5B-GTP and is released in complex with eIF5. eIF5B promotes ribosomal subunit joining, with the help of eIF1A. Upon subunit joining, eIF5B hydrolyzes GTP and is released together with eIF1A. We found that human eIF5 interacts with eIF5B and may help recruit eIF5B to the PIC. An eIF5B-binding motif was identified at the C-terminus of eIF5, similar to that found in eIF1A. Indeed, eIF5 competes with eIF1A for binding and has an ?100-fold higher affinity for eIF5B. Because eIF5 is the GAP of eIF2, the newly discovered interaction offers a possible mechanism for coordination between the two steps in translation initiation controlled by GTPases: start codon selection and ribosomal subunit joining. Our results indicate that in humans, eIF5B displacing eIF2 from Met-tRNAi upon subunit joining may be coupled to eIF1A displacing eIF5 from eIF5B, allowing the eIF5:eIF2-GDP complex to leave the ribosome.