scholarly journals Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo

2007 ◽  
Vol 21 (10) ◽  
pp. 1217-1230 ◽  
Author(s):  
Y.-N. Cheung ◽  
D. Maag ◽  
S. F. Mitchell ◽  
C. A. Fekete ◽  
M. A. Algire ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Start Codon ◽  
40S Ribosomal Subunit ◽  
Codon Selection

scholarly journals eIF3b and eIF3i relocate together to the ribosomal subunit interface during translation initiation and modulate start codon selection

2018 ◽  
Author(s):  
Jose L. Llácer ◽  
Tanweer Hussain ◽  
Jinsheng Dong ◽  
Yuliya Gordiyenko ◽  
Alan G. Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Start Codon ◽  
High Fidelity ◽  
Electron Cryomicroscopy ◽  
Translational Initiation ◽  
Closed Conformation ◽  
Subunit Interface ◽  
Codon Selection

ABSTRACTDuring eukaryotic translational initiation, the 48S ribosomal pre-initiation complex (PIC) scans the 5’ untranslated region of mRNA until it encounters a start codon. We present a single particle electron cryomicroscopy (cryo-EM) reconstruction of a yeast 48S PIC in an open scanning-competent state in which eIF3b is observed bound on the 40S subunit interface. eIF3b is re-located with eIF3i from their solvent-interface locations observed in other PIC structures; however, eIF3i is not in contact with the 40S. Re-processing of micrographs of our previous 48S PIC in a closed state using currently available tools reveal a similar re-location of eIF3b and eIF3i from the solvent to subunit interface. Genetic analysis indicates that high fidelity initiation in vivo depends strongly on eIF3b interactions at the subunit interface that either promote the closed conformation of the PIC on start codon selection or facilitate subsequent relocation back to the solvent side of the 40S subunit.

scholarly journals DHX29 reduces leaky scanning through an upstream AUG codon regardless of its nucleotide context

2016 ◽  
Vol 44 (9) ◽  
pp. 4252-4265 ◽  
Author(s):  
Vera P. Pisareva ◽  
Andrey V. Pisarev
Keyword(s):  
Mutational Analysis ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Start Codon ◽  
Eukaryotic Translation ◽  
Initiation Factors ◽  
40S Ribosomal Subunit ◽  
Nucleotide Context ◽  
Eukaryotic Translation Initiation ◽  
Codon Selection

Abstract 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 eIF2α 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.

scholarly journals Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition

2018 ◽  
Author(s):  
Jose L. Llácer ◽  
Tanweer Hussain ◽  
Adesh K. Saini ◽  
Jagpreet Nanda ◽  
Sukhvir Kaur ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Start Codon ◽  
Dual Function ◽  
Translational Initiation ◽  
Eukaryotic Translation ◽  
Codon Recognition ◽  
40S Ribosomal Subunit

SUMMARYIn 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 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 codons in 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.

scholarly journals Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation

2021 ◽  
Vol 118 (6) ◽  
pp. e2017715118
Author(s):  
Christopher P. Lapointe ◽  
Rosslyn Grosely ◽  
Alex G. Johnson ◽  
Jinfan Wang ◽  
Israel S. Fernández ◽  
...  
Keyword(s):  
Single Molecule ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Start Codon ◽  
Messenger Rnas ◽  
Eukaryotic Translation ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
40S Ribosomal Subunit ◽  
Eukaryotic Translation Initiation Factors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1–40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.

scholarly journals eIF2α interactions with mRNA control accurate start codon selection by the translation preinitiation complex

2020 ◽  
Vol 48 (18) ◽  
pp. 10280-10296
Author(s):  
Anil Thakur ◽  
Swati Gaikwad ◽  
Anil K Vijjamarri ◽  
Alan G Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Ternary Complex ◽  
Start Codon ◽  
Preinitiation Complex ◽  
Closed Conformation ◽  
Open Conformation ◽  
Kozak Context ◽  
Codon Selection

Abstract 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.

scholarly journals The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

2020 ◽  
Author(s):  
Laura Plassart ◽  
Ramtin Shayan ◽  
Christian Montellese ◽  
Dana Rinaldi ◽  
Natacha Larburu ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Inactive Form ◽  
Final Maturation ◽  
Translation Apparatus ◽  
Translation Accuracy ◽  
Functional Analyses

Preventing premature interaction of preribosomes with the translation apparatus is essential to translation accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3′ end, is safeguarded by protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-EM analysis of late human pre-40S particles purified using a catalytically-inactive form of ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATPloaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3′ end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

scholarly journals eIF1 Loop 2 interactions with Met-tRNAi control the accuracy of start codon selection by the scanning preinitiation complex

2018 ◽  
Vol 115 (18) ◽  
pp. E4159-E4168 ◽  
Author(s):  
Anil Thakur ◽  
Alan G. Hinnebusch
Keyword(s):  
Initiation Factor ◽  
Start Codon ◽  
Preinitiation Complex ◽  
Initiation Factor 2 ◽  
D Loop ◽  
Double Substitution ◽  
Codon Selection ◽  
Loop 2

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.

scholarly journals Effects of starvation, diabetes and acute insulin treatment on the regulation of polypeptide-chain initiation in rat skeletal muscle

1984 ◽  
Vol 223 (3) ◽  
pp. 687-696 ◽  
Author(s):  
C S Harmon ◽  
C G Proud ◽  
V M Pain
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Polypeptide Chain ◽  
Ribosomal Subunit ◽  
Diabetic Rats ◽  
Initiation Factor ◽  
Chain Initiation ◽  
Low Concentrations ◽  
40S Ribosomal Subunit

The rate of protein synthesis in skeletal muscle is greatly decreased in response to diabetes and starvation. Analysis of polyribosome profiles indicates that polypeptide-chain initiation is impaired under these conditions. To identify the step in initiation that is affected, we assayed the incorporation of [35S]methionyl-tRNAfMet into [35S]methionyl-tRNAfMet . 40S-ribosomal-subunit initiation complexes in cell-free extracts based on postmitochondrial supernatants prepared from gastrocnemius muscle. Extracts from either starved or diabetic rats were 30-40% less active in forming these complexes compared with those derived from fed or insulin-maintained controls respectively. This change could be reversed by treatment of either starved or diabetic rats with insulin in vivo 30 min before death. Formation of 40S initiation complexes by extracts from either fed or starved rats could be stimulated by the addition of exogenous purified initiation factor eIF-2, but extracts from starved or diabetic rats were more sensitive than controls to stimulation by low concentrations of the factor. These results provide evidence for the acute regulation by insulin of protein synthesis in skeletal muscle at the level of polypeptide-chain initiation, and suggest that in this tissue, as in certain other eukaryotic systems, control of initiation appears to be mediated by changes in the activity of initiation factor eIF-2.

scholarly journals yRACK1/Asc1 proxiOMICs—Towards Illuminating Ships Passing in the Night

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1384 ◽  
Author(s):  
Kerstin Schmitt ◽  
Oliver Valerius
Keyword(s):  
Quality Control ◽  
Binding Proteins ◽  
Ribosomal Subunit ◽  
Stress Factors ◽  
Head Region ◽  
Ubiquitin E3 Ligase ◽  
Mrna Binding Proteins ◽  
40S Ribosomal Subunit ◽  
Mrna Binding

Diverse signals and stress factors regulate the activity and homeostasis of ribosomes in all cells. The Saccharomyces cerevisiae protein Asc1/yRACK1 occupies an exposed site at the head region of the 40S ribosomal subunit (hr40S) and represents a central hub for signaling pathways. Asc1 strongly affects protein phosphorylation and is involved in quality control pathways induced by translation elongation arrest. Therefore, it is important to understand the dynamics of protein formations in the Asc1 microenvironment at the hr40S. We made use of the in vivo protein-proximity labeling technique Biotin IDentification (BioID). Unbiased proxiOMICs from two adjacent perspectives identified nucleocytoplasmic shuttling mRNA-binding proteins, the deubiquitinase complex Ubp3-Bre5, as well as the ubiquitin E3 ligase Hel2 as neighbors of Asc1. We observed Asc1-dependency of hr40S localization of mRNA-binding proteins and the Ubp3 co-factor Bre5. Hel2 and Ubp3-Bre5 are described to balance the mono-ubiquitination of Rps3 (uS3) during ribosome quality control. Here, we show that the absence of Asc1 resulted in massive exposure and accessibility of the C-terminal tail of its ribosomal neighbor Rps3 (uS3). Asc1 and some of its direct neighbors together might form a ribosomal decision tree that is tightly connected to close-by signaling modules.

scholarly journals The methyltransferase adaptor protein Trm112 is involved in biogenesis of both ribosomal subunits

2012 ◽  
Vol 23 (21) ◽  
pp. 4313-4322 ◽  
Author(s):  
Richa Sardana ◽  
Arlen W. Johnson
Keyword(s):  
Slow Growth ◽  
Ribosomal Subunit ◽  
Adaptor Protein ◽  
Small Subunit ◽  
Stable Complex ◽  
Sedimentation Analysis ◽  
Ribosomal Subunits ◽  
Translation Factors ◽  
40S Ribosomal Subunit

We previously identified Bud23 as the methyltransferase that methylates G1575 of rRNA in the P-site of the small (40S) ribosomal subunit. In this paper, we show that Bud23 requires the methyltransferase adaptor protein Trm112 for stability in vivo. Deletion of Trm112 results in a bud23Δ-like mutant phenotype. Thus Trm112 is required for efficient small-subunit biogenesis. Genetic analysis suggests the slow growth of a trm112Δ mutant is due primarily to the loss of Bud23. Surprisingly, suppression of the bud23Δ-dependent 40S defect revealed a large (60S) biogenesis defect in a trm112Δ mutant. Using sucrose gradient sedimentation analysis and coimmunoprecipitation, we show that Trm112 is also involved in 60S subunit biogenesis. The 60S defect may be dependent on Nop2 and Rcm1, two additional Trm112 interactors that we identify. Our work extends the known range of Trm112 function from modification of tRNAs and translation factors to both ribosomal subunits, showing that its effects span all aspects of the translation machinery. Although Trm112 is required for Bud23 stability, our results suggest that Trm112 is not maintained in a stable complex with Bud23. We suggest that Trm112 stabilizes its free methyltransferase partners not engaged with substrate and/or helps to deliver its methyltransferase partners to their substrates.

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