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The histone acetyltransferase (HAT) subunit of coactivator complex SAGA, Gcn5, stimulates eviction of promoter nucleosomes at certain highly expressed yeast genes, including those activated by transcription factor Gcn4 in amino acid-deprived cells; however, the importance of other HAT complexes in this process was poorly understood. Analyzing mutations that disrupt the integrity or activity of HAT complexes NuA4 or NuA3, or HAT Rtt109, revealed that only NuA4 acts on par with Gcn5, and functions additively, in evicting and repositioning promoter nucleosomes and stimulating transcription of starvation-induced genes. NuA4 is generally more important than Gcn5, however, in promoter nucleosome eviction, TBP recruitment, and transcription at most other genes expressed constitutively. NuA4 also predominates over Gcn5 in stimulating TBP recruitment and transcription of genes categorized as principally dependent on the cofactor TFIID versus SAGA, except for the most highly expressed subset including ribosomal protein genes, where Gcn5 contributes strongly to PIC assembly and transcription. Both SAGA and NuA4 are recruited to promoter regions of starvation-induced genes in a manner that might be feedback controlled by their HAT activities. Our findings reveal an intricate interplay between these two HATs in nucleosome eviction, PIC assembly, and transcription that differs between the starvation-induced and basal transcriptomes.

The General Amino Acid Control is a conserved response to amino acid starvation involving activation of protein kinase Gcn2, which phosphorylates eukaryotic initiation factor 2 (eIF2α) with attendant inhibition of global protein synthesis and increased translation of yeast transcriptional activator GCN4 . Gcn2 can be activated by either amino acid starvation or conditions that stall elongating ribosomes without reducing aminoacylation of tRNA, but it is unclear whether distinct molecular mechanisms operate in these two circumstances. We identified three regimes that activate Gcn2 in yeast cells by starvation-independent (SI) ribosome-stalling: treatment with tigecycline, eliminating the sole gene encoding tRNA Arg UCC , and depletion of translation termination factor eRF1. We further demonstrated requirements for the tRNA- and ribosome-binding domains of Gcn2, the positive effector proteins Gcn1/Gcn20, and the tethering of at least one of two distinct P1/P2 heterodimers to the uL10 subunit of the ribosomal P stalk, for detectable activation by SI-ribosome stalling. Remarkably, no tethered P1/P2 proteins were required for strong Gcn2 activation elicited by starvation for histidine or branched-chain amino acids isoleucine/valine. These results indicate that Gcn2 activation has different requirements for the P stalk depending on how ribosomes are stalled. We propose that accumulation of deacylated tRNAs in amino acid-starved cells can functionally substitute for the P stalk in binding to the histidyl-tRNA synthetase-like domain of Gcn2 for eIF2α kinase activation by ribosomes stalled with A sites devoid of the eEF1A∙GTP∙aminoacyl–tRNA ternary complex.

21 páginas, 7 figuras, 2 tablas. Funding for open access charge: Consejo Superior de Investigaciones Científicas/SpanishNational Research Council, The eukaryotic initiation factor 3 (eIF3) complex is involved in every step of translation initiation, but there is limited understanding of its molecular functions. Here, we present a single particle electron cryomicroscopy (cryo-EM) reconstruction of yeast 48S ribosomal preinitiation complex (PIC) in an open conformation conducive to scanning, with core subunit eIF3b bound on the 40S interface near the decoding center in contact with the ternary complex eIF2·GTP·initiator tRNA. eIF3b is relocated together with eIF3i from their solvent interface locations observed in other PIC structures, with eIF3i lacking 40S contacts. Re-processing of micrographs of our previous 48S PIC in a closed state also suggests relocation of the entire eIF3b-3i-3g-3a-Cter module during the course of initiation. Genetic analysis indicates that high fidelity initiation depends on eIF3b interactions at the 40S subunit interface that promote the closed PIC conformation, or facilitate the relocation of eIF3b/eIF3i to the solvent interface, on start codon selection., Spanish government [BFU2017-85814-P]; Generalitat Valenciana [SEJI/2019/030 to J.L.L.]; start-up funds [R(IV)090/1076/2017-4252] from the Indian Institute of Science, Bangalore, India; Wellcome Trust/DBT India Alliance Fellowship [IA/I/17/2/503313 to T.H.]; Human Frontiers in Science Program [RGP-0028/2009]; Intramural Research Program of the NIH (to A.G.H.); T.H. also thanks DST-FIST [SR/FST/LS11-036/2014(C)], UGCSAP [F.4.13/2018/DRS-III (SAP-II)]; DBT-IISc Partnership Program Phase-II [BT/PR27952-NF/22/212/2018] for infrastructure and financial support. Funding for open access charge: Consejo Superior de Investigaciones Cient´ıficas/SpanishNational Research Council.

The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that nonlethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.

BackgroundTranslation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as UUG, ACG and AUU are also used as start sites at low levels inS. cerevisiae. Initiation from NCCs or AUGs in the 5’-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature.ResultsUsing reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs inS. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 °C relative to 30 °C and decreased efficiency at 20 °C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5’-leader relative to the 5’-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation.ConclusionsOverall, our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression inS. cerevisiae.

In eukaryotes, 43S preinitiation complex (PIC) formation is a rate-determining step of translation. Ribosome recycling following translation termination produces free 40S subunits for re-assembly of 43S PICs. Yeast mutants lacking orthologs of mammalian eIF2D (Tma64), and either MCT-1 (Tma20) or DENR (Tma22), are broadly impaired for 40S recycling; however, it was unknown whether this defect alters the translational efficiencies (TEs) of particular mRNAs. Here, we conducted ribosome profiling of a yeast tma64∆/tma20∆ double mutant and observed a marked reprogramming of translation, wherein the TEs of the most efficiently translated (‘strong’) mRNAs increase, while those of ‘weak’ mRNAs generally decline. Remarkably, similar reprogramming was seen on reducing 43S PIC assembly by inducing phosphorylation of eIF2α or by decreasing total 40S subunit levels by depleting Rps26. Our findings suggest that strong mRNAs outcompete weak mRNAs in response to 43S PIC limitation achieved in various ways, in accordance with previous mathematical modeling.

The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the −1 and +1 nucleosomes flanking NDRs; however, Ino80C’s function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the −1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.

In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the −3 nucleotide of the ‘Kozak’ context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui− cells (Ssu− phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui− phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd− phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.

Living cells have developed exquisite mechanisms to ensure accurate translation of mRNA. Many of them are dedicated to preventing the change in reading frame during translation elongation. A minority of chromosomally encoded genes have evolved sequences that subvert standard decoding to program +1 translational frameshifting, either constitutively or in response to external stimuli. In the yeastSaccharomyces cerevisiae, three chromosomal genes are known to employ programmed +1 translational frameshifting for expression of full-length functional products. Here we identify a fourth yeast gene,YFS1, encompassing the existing predicted open reading frameYPL034W, with conserved programmed +1 frameshifting. Like the previously known examples, it appears to exploit peculiarities in tRNA abundance inS. cerevisiae.

Yeast DEAD-box helicase Ded1 stimulates translation initiation, particularly of mRNAs with structured 5'UTRs. Interactions of the Ded1 N-terminal domain (NTD) with eIF4A, and Ded1-CTD with eIF4G, subunits of eIF4F, enhance Ded1 unwinding activity and stimulation of preinitiation complex (PIC) assembly in vitro. However, the importance of these interactions, and of Ded1-eIF4E association, in vivo were poorly understood. We identified separate amino acid clusters in the Ded1-NTD required for binding to eIF4A or eIF4E in vitro. Disrupting each cluster selectively impairs native Ded1 association with eIF4A or eIF4E, and reduces cell growth, polysome assembly, and translation of reporter mRNAs with structured 5'UTRs. It also impairs Ded1 stimulation of PIC assembly on a structured mRNA in vitro. Ablating Ded1 interactions with eIF4A/eIF4E unveiled a requirement for the Ded1-CTD for robust initiation. Thus, Ded1 function in vivo is stimulated by independent interactions of its NTD with eIF4E and eIF4A, and its CTD with eIF4G.

In translation initiation, a 43S preinitiation complex (PIC) containing eIF1 and a ternary complex (TC) of GTP-bound eIF2 and Met-RNAi scans the mRNA for the start codon. AUG recognition triggers eIF1 release and rearrangement from an open PIC conformation to a closed state with more tightly-bound Met-tRNAi (PIN state). Cryo-EM models reveal eIF2β contacts with eIF1 and Met-tRNAi exclusive to the open complex that should destabilize the closed state. eIF2β or eIF1 substitutions disrupting these contacts increase initiation at UUG codons, and compound substitutions also derepress translation of GCN4, indicating slower TC recruitment. The latter substitutions slow TC loading while stabilizing TC binding at UUG codons in reconstituted PICs, indicating a destabilized open complex and shift to the closed/PIN state. An eIF1 substitution that should strengthen the eIF2β:eIF1 interface has the opposite genetic and biochemical phenotypes. eIF2β is also predicted to restrict Met-tRNAi movement into the closed/PIN state, and substitutions that should diminish this clash increase UUG initiation in vivo and stabilize Met-tRNAi binding at UUG codons in vitro with little effect on TC loading. Thus, eIF2β anchors eIF1 and TC to the open complex, enhancing PIC assembly and scanning, while impeding rearrangement to the closed conformation at non-AUG codons.

The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of −1 and +1 nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by transcription factor Gcn4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of Gcn4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs—highly depleted of nucleosomes—during transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.

Protein production must be strictly controlled at its beginning and end to synthesize a polypeptide that faithfully copies genetic information carried in the encoding mRNA. In contrast to viruses and prokaryotes, the majority of mRNAs in eukaryotes contain only one coding sequence, resulting in production of a single protein. There are, however, many exceptional mRNAs that either carry short open reading frames upstream of the main coding sequence (uORFs) or even contain multiple long ORFs. A wide variety of mechanisms have evolved in microbes and higher eukaryotes to prevent recycling of some or all translational components upon termination of the first translated ORF in such mRNAs and thereby enable subsequent translation of the next uORF or downstream coding sequence. These specialized reinitiation mechanisms are often regulated to couple translation of the downstream ORF to various stimuli. Here we review all known instances of both short uORF-mediated and long ORF-mediated reinitiation and present our current understanding of the underlying molecular mechanisms of these intriguing modes of translational control., This review covers one of many means of gene-specific translational control called translational reinitiation that (i) occurs on mRNAs carrying more than one ORF, (ii) can be mediated by either post-termination 40S or 70S/80S ribosomes, and (iii) is highly regulated in response to various stresses and many other intra- or extracellular signals.

Initiation of translation on eukaryotic mRNAs generally follows the scanning mechanism, wherein a preinitiation complex (PIC) assembled on the small (40S) ribosomal subunit and containing initiator methionyl tRNAi (Met-tRNAi) scans the mRNA leader for an AUG codon. In a current model, the scanning PIC adopts an open conformation and rearranges to a closed state, with fully accommodated Met-tRNAi, upon AUG recognition. Evidence from recent high-resolution structures of PICs assembled with different ligands supports this model and illuminates the molecular functions of eukaryotic initiation factors eIF1, eIF1A, and eIF2 in restricting to AUG codons the transition to the closed conformation. They also reveal that the eIF3 complex interacts with multiple functional sites in the PIC, rationalizing its participation in numerous steps of initiation.

SUMMARYTranslational control targeting mainly the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast Translational Complex Profile sequencing (TCP-seq), related to ribosome profiling, and adopted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ses) in addition to 80S ribosomes (80Ses), revealed that mammalian and yeast 40Ses distribute similarly across 5’UTRs indicating considerable evolutionary conservation. We further developed a variation called Selective TCP-seq (Sel-TCP-seq) enabling selection for 40Ses and 80Ses associated with an immuno-targeted factor in yeast and human. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5’UTRs with scanning 40Ses to successively dissociate upon start codon recognition. Manifesting the Sel-TCP-seq versatility for gene expression studies, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

The translation preinitiation complex (PIC) scans the mRNA for an AUG codon in a favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with the reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at certain NCCs initiating amino-terminal extensions, including those that direct mitochondrial localization of the GRS1 and ALA1 products, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.

RNA structures that impede ribosome binding or subsequent scanning of the 5′-untranslated region (5′-UTR) for the AUG initiation codon reduce translation efficiency. Yeast DEAD-box RNA helicase Ded1 appears to promote translation by resolving 5′-UTR structures, but whether its paralog, Dbp1, performs similar functions is unknown. Furthermore, direct in vivo evidence was lacking that Ded1 or Dbp1 resolves 5′-UTR structures that impede attachment of the 43S preinitiation complex (PIC) or scanning. Here, profiling of translating 80S ribosomes reveals that the translational efficiencies of many more mRNAs are reduced in a ded1-ts dbp1Δ double mutant versus either single mutant, becoming highly dependent on Dbp1 or Ded1 only when the other helicase is impaired. Such ‘conditionally hyperdependent’ mRNAs contain unusually long 5′-UTRs with heightened propensity for secondary structure and longer transcript lengths. Consistently, overexpressing Dbp1 in ded1 cells improves the translation of many such Ded1-hyperdependent mRNAs. Importantly, Dbp1 mimics Ded1 in conferring greater acceleration of 48S PIC assembly in a purified system on mRNAs harboring structured 5′-UTRs. Profiling 40S initiation complexes in ded1 and dbp1 mutants provides direct evidence that Ded1 and Dbp1 cooperate to stimulate both PIC attachment and scanning on many Ded1/Dbp1-hyperdependent mRNAs in vivo.

Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.

DEAD-box RNA helicases eukaryotic translation initiation factor 4A (eIF4A) and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. Eukaryotic translation initiation factor 4B (eIF4B) is a cofactor for eIF4A but also might function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5' untranslated regions (UTRs). These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop messenger ribonucleoprotein particles (mRNPs) via eIF4F-poly(A)-binding protein 1 (Pab1) association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eukaryotic translation initiation factor 4G (eIF4G), the scaffold subunit of eukaryotic translation initiation factor 4F (eIF4F), preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5' UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly.

The eukaryotic 5′ untranslated region (UTR) is critical for ribosome recruitment to the messenger RNA (mRNA) and start codon choice and plays a major role in the control of translation efficiency and shaping the cellular proteome. The ribosomal initiation complex is assembled on the mRNA via a cap-dependent or cap-independent mechanism. We describe various mechanisms controlling ribosome scanning and initiation codon selection by 5′ upstream open reading frames, translation initiation factors, and primary and secondary structures of the 5′UTR, including particular sequence motifs. We also discuss translational control via phosphorylation of eukaryotic initiation factor 2, which is implicated in learning and memory, neurodegenerative diseases, and cancer.

Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4., Author summary Previous work showed that Scd6 homologs in Drosophila, Xenopus, and humans are associated with translationally repressed mRNAs in RNA granules, and that they interact with other mRNA silencing factors, including homologs of RNA helicase Dhh1. However, there is little evidence that such Scd6 homologs are critical for translational repression or degradation of specific mRNAs in vivo. Yeast Scd6 interacts with the mRNA decapping enzyme and was shown to inhibit translation in cell extracts through binding to cap-binding translation initiation factor eIF4G. However, it was unknown whether Scd6 represses translation or accelerates degradation of any specific mRNAs in vivo, or whether Scd6 requires the decapping enzyme or its activators for such events. Here we show that tethering Scd6 stimulates the degradation or translational repression of reporter mRNAs in yeast, collaborating with Dhh1 for translational repression and the decapping enzyme Dcp2/Dcp1 for mRNA turnover. Using ribosome profiling we further identify groups of native mRNAs that appear to be targeted for degradation or translational repression by Scd6 and Dhh1 and find unexpectedly that Dcp2 is necessary for translational repression as well as enhanced degradation of most such mRNAs. Thus, Scd6 partners with Dhh1 and Dcp2 to mediate translational repression and degradation of specific mRNAs in vivo.

During translational initiation in eukaryotes, the 40S ribosomal subunit forms a 48S pre-initiation complex (PIC) with eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAMeti) and eIF5. The 48S PIC, in an open conformation, scans the 5' untranslated region of mRNA until it encounters a start codon in the P site. We present a single particle cryo-electron microscopy (cryo-EM) reconstruction of a 48S PICs from yeast in an open scanning-competent state at 5.15[A] resolution. The structure contains eIF3 PCI domains bound on the solvent side of the 40S, that was not observed in the earlier reported complex in an open state. eIF3b is observed bound on the 40S subunit interface, re-located with eIF3i from their solvent-interface locations observed in other PIC structures; however, the eIF3i {beta}-propeller is not in contact with the 40S. We also present a map obtained by masked classification with signal subtraction around the observed {beta}-propeller of eIF3b on the intersubunit interface of our previous 48S PICs in closed state, which also shows similar re-location of eIF3b and eIF3i from the solvent interface. Overall, the structures reported here are similar to those reported earlier but the relatively higher resolution of 48S PIC-open and the higher local resolution around the eIF3 {beta}-propellers in py48S-closed enabled a more detailed assignment of eIF3 subunits and revealed relocation of the entire quaternary complex of eIF3b/eIF3i/eIF3g/eIF3a-Cter to the intersubunit interface of 40S.

33 páginas, 10 figuras, 5 tablas, In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNAi in a 'PIN' state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG codonsin vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection., We are grateful to CG Savva, G McMullan for technical support with cryo-EM, T Darling and J Grimmett for help with computing and J Brasa for help with figures/movies. JL was supported by a FEBS postdoctoral fellowship. TH acknowledges start-up funds from the Indian Institute of Science. This work was funded by grants to from the UK Medical Research Council (MC_U105184332), Wellcome Trust Senior Investigator award (WT096570), the Agouron Institute and the Jeantet Foundation to VR; by the Department of Science and Technology, Government of India Grant [Int/NZ/P-2/13] to AKS; from the NIH (GM62128) formerly to JRL; the Human Frontiers in Science Program (RGP-0028/ 2009) to AGH, JRL and VR; and by the Intramural Research Program of the NIH (AGH, JRL).

In translation initiation, a preinitiation complex (PIC) of the 40S ribosomal subunit, eukaryotic initiation factors (eIFs), and initiator transfer RNA (tRNAi) scans the mRNA for an AUG codon in favorable context. AUG recognition evokes a closed PIC conformation and triggers dissociation of eIF1. Structural data predict that a clash between eIF1 β-hairpin Loop 2 and tRNAi impedes selection of non-AUG start codons in the open, scanning conformation of the PIC. Supporting this model, we show that alanine substitutions of Loop 2 residues increase initiation at suboptimal start codons (UUGs and AUGs in poor context) in vivo and destabilize tRNAi binding at UUG codons in reconstituted PICs in vitro. These results establish Loop 2–RNAi interaction as a key determinant of initiation accuracy.

The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNAi) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a “POUT” state to a closed conformation with TC more tightly bound in a “PIN” state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the PIN state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNAi D loop in the PIN state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNAi. We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2–D loop interactions specifically impede Met-tRNAi accommodation in the PIN state without influencing the POUT mode of TC binding; and Arg substitutions convert the Loop 2–tRNAi clash to an electrostatic attraction that stabilizes PIN and enhances selection of poor start codons in vivo.

Gcn4 is a yeast transcriptional activator induced by amino acid starvation. ChIP-seq analysis revealed 546 genomic sites occupied by Gcn4 in starved cells, representing ∼30% of Gcn4-binding motifs. Surprisingly, only ∼40% of the bound sites are in promoters, of which only ∼60% activate transcription, indicating extensive negative control over Gcn4 function. Most of the remaining ∼300 Gcn4-bound sites are within coding sequences (CDSs), with ∼75 representing the only bound sites near Gcn4-induced genes. Many such unconventional sites map between divergent antisense and sub-genic sense transcripts induced within CDSs adjacent to induced TBP peaks, consistent with Gcn4 activation of cryptic bidirectional internal promoters. Mutational analysis confirms that Gcn4 sites within CDSs can activate sub-genic and full-length transcripts from the same or adjacent genes, showing that functional Gcn4 binding is not confined to promoters. Our results show that internal promoters can be regulated by an activator that functions at conventional 5'-positioned promoters.

Chaperones, nucleosome remodeling complexes, and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these cofactors function ubiquitously, as well as the impact of nucleosome eviction on transcription genome-wide, is poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple cofactors to address these issues for about 200 genes belonging to the Gcn4 transcriptome, of which about 70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 cochaperone Ydj1, and chromatin-associated factor Yta7 are required downstream from Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC, and H2AZ are dispensable for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double, and triple mutants implicated Gcn5, Snf2, and Ydj1 in H3 eviction at most, but not all, Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these three cofactors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in cofactor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes but that other steps in gene activation are more rate-limiting for most other yeast genes.

SummaryTo study the function of Rli1/ABCE1 in vivo, we used ribosome profiling and biochemistry to characterize its contribution to ribosome recycling. When Rli1 levels were diminished, 80S ribosomes accumulated both at stop codons and in the adjoining 3′UTRs of most mRNAs. Frequently, these ribosomes reinitiated translation without the need for a canonical start codon, as small peptide products predicted by 3′UTR ribosome occupancy in all three reading frames were confirmed by western analysis and mass spectrometry. Eliminating the ribosome-rescue factor Dom34 dramatically increased 3′UTR ribosome occupancy in Rli1 depleted cells, indicating that Dom34 clears the bulk of unrecycled ribosomes. Thus, Rli1 is crucial for ribosome recycling in vivo and controls ribosome homeostasis. 3′UTR translation occurs in wild-type cells as well, and observations of elevated 3′UTR ribosomes during stress suggest that modulating recycling and reinitiation is involved in responding to environmental changes.

Summary Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition., Graphical Abstract, Highlights • Structures of eukaryotic translation initiation complexes in open and closed states • In the open complex the 40S head moves upward to open the mRNA entry channel latch • Transition to closed state locks initiator tRNA in the P site base-paired with AUG • The structures show how eIF3 contacts eIF2 and eIF1 on the 40S subunit interface, The small ribosomal subunit latch has to open to allow mRNA loading. Llacer et al. report cryo-EM reconstructions of yeast translation pre-initiation complexes in both open and closed conformations, highlighting structural differences and elucidating key events in start codon recognition.

DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5′ end or subsequent scanning of the 5′ UTR, but whether they perform unique or overlapping functions in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in budding yeast Saccharomyces cerevisiae by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A variant encoded by tif1-A79V (in a strain lacking the ortholog TIF2) yielded ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5′ UTR length and propensity for secondary structure, implicating Ded1 in scanning through structured 5′ UTRs. Reporter assays confirmed that cap-distal stem–loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs shows a heightened requirement for eIF4A, dependence on eIF4A is correlated with requirements for Ded1 and 5′ UTR features characteristic of Ded1-dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5′ UTRs, and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs.

The last 20 years have seen tremendous progress in deciphering the fundamental mechanisms of protein synthesis and molecular pathways of translational control, and in implicating these processes in development, neurobiology, and etiologyof human diseases. Muchof this success can be attributed to the introduction of new technologies, such as next-generation RNA sequencing and advances in cryo-EM; and also to development of powerful in vitro systems recapitulating complex in vivo phenomena with purified components. Also critical have been strategic interactions between experts in different disciplines of biology, such as the wedding of biochemistry with genetics and structural biology, and of translation biochemistry with neurobiology and medicine. RNA has contributed to this progress by publishing key findings on molecular mechanisms of protein synthesis and the role of translational control in regulating gene expression in diverse organisms. Thebasic reactions of the elongationand termination stages of protein synthesis are highly conserved throughout all three kingdoms of life. Because they occur in the catalytic center of the ribosome, their molecular mechanisms were illuminated by the stunning successes of X-ray crystallography and cryo-electron microscopy in producing high-resolution structures of both empty ribosomes and a myriad of ribosome complexes with mRNA, tRNAs, or elongation, termination, or ribosome recycling factors trapped in different stages of the process. Recent innovations in cryo-EM technology are revealing ribosome structures at nearly 3 A resolution; and new algorithms for sorting structurally distinct molecules means that sample heterogeneity (an anathema to crystallography) is now exploited to identify different conformational states in the same preparation. The solved ribosome structures revealed the path of mRNA and the A, P, and E binding sites for the tRNA, as it progress through the elongation cycle from aminoacyltRNA, to peptidyl-tRNA to deacylated tRNA, located at the interface between the large and small subunits; as well as the peptidyl transferase center wherepeptide bond formation occurs. As the decoding center of the ribosome is comprised almost entirely of rRNA, it became clear that the ribosome is a ribozyme. The protein factors involved in translocation of mRNA and the tRNAs through the decoding sites (EF-G/ EF2), in recognizing stop codons and hydrolyzing peptidyltRNA (RF1/eRF1), and splitting apart ribosomal subunits following termination (bacterial RRF) all exploit striking structural mimicry of tRNA to fit into the A site. Interestingly, RF1 was shown to contain a protein element that “reads” the stop codon and another that reaches into the peptidyl transferase center to hydrolyze the peptidyl-tRNA linkage. The peptidyl-tRNA and aminoacyl-tRNA are anchored in the precise locations required for peptide bond formation, but the ribosome is not simply a rigid catalytic platform, but undergoes conformational changes in selecting the correct EF1A·GTP·aminoacyl-tRNA ternary complex for each codon, and displays inter-subunit ratcheting during translocation and termination. GTP binding and hydrolysis by the elongation factors evoke cyclic conformational changes that drive the process forward. The use of fluorescently labeled reactants and stopped-flow kinetic analysis allowed a detailed dissection of different steps in the processes of aminoacyltRNA selection in the A site, with proofreading of the codon:anticodon interaction; and of translocation of tRNAs through the decoding center catalyzed by EF2. Single-molecule approaches also contributed importantly to this progress, by identifying intermediate states too transient to be detected by ensemble kinetic experiments or structural biology. In other developments, eIF5A and its bacterial counterpart EF-P were shown to stimulate elongation specifically through stretches of proline codons, and both factors occupy the E-site and communicate directly with the peptidyl transferase center. Interestingly, RRF is replaced in eukaryotes and archaea by an ATP-binding cassette protein, ABCE1/ Rli1, that becomes wedged between the large and small subunits and uses ATP hydrolysis to split apart the subunits. ABCE1/Rli1also functions with RF1 paralog Dom34 to