scholarly journals Initiation factor 2 stabilizes the ribosome in a semirotated conformation

2015 ◽  
Vol 112 (52) ◽  
pp. 15874-15879 ◽  
Author(s):  
Clarence Ling ◽  
Dmitri N. Ermolenko
Keyword(s):  
Single Molecule ◽  
Translational Control ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Bound State ◽  
Initiation Factor ◽  
Large Ribosomal Subunit ◽  
Control Of Gene Expression ◽  
Initiation Phase ◽  
Intersubunit Rotation

Intersubunit rotation and movement of the L1 stalk, a mobile domain of the large ribosomal subunit, have been shown to accompany the elongation cycle of translation. The initiation phase of protein synthesis is crucial for translational control of gene expression; however, in contrast to elongation, little is known about the conformational rearrangements of the ribosome during initiation. Bacterial initiation factors (IFs) 1, 2, and 3 mediate the binding of initiator tRNA and mRNA to the small ribosomal subunit to form the initiation complex, which subsequently associates with the large subunit by a poorly understood mechanism. Here, we use single-molecule FRET to monitor intersubunit rotation and the inward/outward movement of the L1 stalk of the large ribosomal subunit during the subunit-joining step of translation initiation. We show that, on subunit association, the ribosome adopts a distinct conformation in which the ribosomal subunits are in a semirotated orientation and the L1 stalk is positioned in a half-closed state. The formation of the semirotated intermediate requires the presence of an aminoacylated initiator, fMet-tRNAfMet, and IF2 in the GTP-bound state. GTP hydrolysis by IF2 induces opening of the L1 stalk and the transition to the nonrotated conformation of the ribosome. Our results suggest that positioning subunits in a semirotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding.

4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle

2000 ◽  
Vol 279 (4) ◽  
pp. E715-E729 ◽  
Author(s):  
O. Jameel Shah ◽  
Joshua C. Anthony ◽  
Scot R. Kimball ◽  
Leonard S. Jefferson
Keyword(s):  
Translational Control ◽  
Mrna Translation ◽  
Cellular Protein ◽  
Initiation Factor ◽  
Translational Repressor ◽  
Initiation Factors ◽  
Initiation Phase ◽  
Eukaryotic Initiation Factor 4E ◽  
Integration Sites ◽  
A Site

Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesis-degradation equilibrium that is conditionally modulated under an extensive range of physiological and pathophysiological circumstances. Recent studies have established the initiation phase of mRNA translation as a pivotal site of regulation for global rates of protein synthesis, as well as a site through which the synthesis of specific proteins is controlled. The protein synthetic pathway is exquisitely sensitive to the availability of hormones and nutrients and employs a comprehensive integrative strategy to interpret the information provided by hormonal and nutritional cues. The translational repressor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1) have emerged as important components of this strategy, and together they coordinate the behavior of both eukaryotic initiation factors and the ribosome. This review discusses the role of 4E-BP1 and S6K1 in translational control and outlines the mechanisms through which hormones and nutrients effect changes in mRNA translation through the influence of these translational effectors.

scholarly journals A Complementary Mechanism of Bacterial mRNA Translation Inhibition by Tetracyclines

2021 ◽  
Vol 12 ◽  
Author(s):  
Victor Barrenechea ◽  
Maryhory Vargas-Reyes ◽  
Miguel Quiliano ◽  
Pohl Milón
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Resistance Mechanisms ◽  
Dissociation Rate ◽  
Initiation Factor ◽  
Translation Elongation ◽  
Initiation Phase ◽  
Complementary Mechanism

Tetracycline has positively impacted human health as well as the farming and animal industries. Its extensive usage and versatility led to the spread of resistance mechanisms followed by the development of new variants of the antibiotic. Tetracyclines inhibit bacterial growth by impeding the binding of elongator tRNAs to the ribosome. However, a small number of reports indicated that Tetracyclines could also inhibit translation initiation, yet the molecular mechanism remained unknown. Here, we use biochemical and computational methods to study how Oxytetracycline (Otc), Demeclocycline (Dem), and Tigecycline (Tig) affect the translation initiation phase of protein synthesis. Our results show that all three Tetracyclines induce Initiation Factor IF3 to adopt a compact conformation on the 30S ribosomal subunit, similar to that induced by Initiation Factor IF1. This compaction was faster for Tig than Dem or Otc. Furthermore, all three tested tetracyclines affected IF1-bound 30S complexes. The dissociation rate constant of IF1 in early 30S complexes was 14-fold slower for Tig than Dem or Otc. Late 30S initiation complexes (30S pre-IC or IC) exhibited greater IF1 stabilization by Tig than for Dem and Otc. Tig and Otc delayed 50S joining to 30S initiation complexes (30S ICs). Remarkably, the presence of Tig considerably slowed the progression to translation elongation and retained IF1 in the resulting 70S initiation complex (70S IC). Molecular modeling of Tetracyclines bound to the 30S pre-IC and 30S IC indicated that the antibiotics binding site topography fluctuates along the initiation pathway. Mainly, 30S complexes show potential contacts between Dem or Tig with IF1, providing a structural rationale for the enhanced affinity of the antibiotics in the presence of the factor. Altogether, our data indicate that Tetracyclines inhibit translation initiation by allosterically perturbing the IF3 layout on the 30S, retaining IF1 during 70S IC formation, and slowing the transition toward translation elongation. Thus, this study describes a new complementary mechanism by which Tetracyclines may inhibit bacterial protein synthesis.

scholarly journals A Retrospective on eIF2A—and Not the Alpha Subunit of eIF2

2020 ◽  
Vol 21 (6) ◽  
pp. 2054
Author(s):  
Anton A. Komar ◽  
William C. Merrick
Keyword(s):  
Translational Control ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Single Chain ◽  
Initiation Factors ◽  
Eif2α Phosphorylation ◽  
Specific Mrnas ◽  
Internal Initiation ◽  
And Function ◽  
Polypeptide Chains

Initiation of protein synthesis in eukaryotes is a complex process requiring more than 12 different initiation factors, comprising over 30 polypeptide chains. The functions of many of these factors have been established in great detail; however, the precise role of some of them and their mechanism of action is still not well understood. Eukaryotic initiation factor 2A (eIF2A) is a single chain 65 kDa protein that was initially believed to serve as the functional homologue of prokaryotic IF2, since eIF2A and IF2 catalyze biochemically similar reactions, i.e., they stimulate initiator Met-tRNAi binding to the small ribosomal subunit. However, subsequent identification of a heterotrimeric 126 kDa factor, eIF2 (α,β,γ) showed that this factor, and not eIF2A, was primarily responsible for the binding of Met-tRNAi to 40S subunit in eukaryotes. It was found however, that eIF2A can promote recruitment of Met-tRNAi to 40S/mRNA complexes under conditions of inhibition of eIF2 activity (eIF2α-phosphorylation), or its absence. eIF2A does not function in major steps in the initiation process, but is suggested to act at some minor/alternative initiation events such as re-initiation, internal initiation, or non-AUG initiation, important for translational control of specific mRNAs. This review summarizes our current understanding of the eIF2A structure and function.

scholarly journals Neuronal BC RNAs cooperate with eIF4B to mediate activity-dependent translational control

2014 ◽  
Vol 207 (2) ◽  
pp. 237-252 ◽  
Author(s):  
Taesun Eom ◽  
Ilham A. Muslimov ◽  
Panayiotis Tsokas ◽  
Valerio Berardi ◽  
Jun Zhong ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Translational Control ◽  
Translational Regulation ◽  
Regulation Of Gene Expression ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Neuronal Stimulation ◽  
Mechanistic Basis ◽  
Protein Kinase Mζ ◽  
Activity Dependent

In neurons, translational regulation of gene expression has been implicated in the activity-dependent management of synapto-dendritic protein repertoires. However, the fundamentals of stimulus-modulated translational control in neurons remain poorly understood. Here we describe a mechanism in which regulatory brain cytoplasmic (BC) RNAs cooperate with eukaryotic initiation factor 4B (eIF4B) to control translation in a manner that is responsive to neuronal activity. eIF4B is required for the translation of mRNAs with structured 5′ untranslated regions (UTRs), exemplified here by neuronal protein kinase Mζ (PKMζ) mRNA. Upon neuronal stimulation, synapto-dendritic eIF4B is dephosphorylated at serine 406 in a rapid process that is mediated by protein phosphatase 2A. Such dephosphorylation causes a significant decrease in the binding affinity between eIF4B and BC RNA translational repressors, enabling the factor to engage the 40S small ribosomal subunit for translation initiation. BC RNA translational control, mediated via eIF4B phosphorylation status, couples neuronal activity to translational output, and thus provides a mechanistic basis for long-term plastic changes in nerve cells.

Contrasting mechanisms of regulating translation of specific Drosophila germline mRNAs at the level of 5′-cap structure binding

2005 ◽  
Vol 33 (6) ◽  
pp. 1544-1546 ◽  
Author(s):  
P. Lasko ◽  
P. Cho ◽  
F. Poulin ◽  
N. Sonenberg
Keyword(s):  
Translational Control ◽  
Binding Proteins ◽  
Regulatory Mechanism ◽  
Ribosomal Subunit ◽  
Drosophila Embryo ◽  
Initiation Factor ◽  
Control Mechanisms ◽  
Eukaryotic Initiation Factor ◽  
Eukaryotic Initiation Factor 4E ◽  
Cognate Protein

Translational control is a key genetic regulatory mechanism underlying the initial establishment of the major spatial axes of the Drosophila embryo. Many translational control mechanisms target eIF4E (eukaryotic initiation factor 4E), an initiation factor that recognizes the 5′-cap structure of the mRNA. Cap recognition by eIF4E, in complex with eIF4G, is essential for recruitment of the mRNA to the small ribosomal subunit. One established mechanism for repressing translation involves eIF4E-binding proteins, which competitively inhibit the eIF4E–eIF4G interaction. Our group has uncovered a novel mechanism for repression in which an eIF4E cognate protein called d4EHP, which cannot bind eIF4G, binds to the 5′-cap structure of cad mRNA thus rendering it translationally inactive. These two related, but distinct, mechanisms are discussed and contrasted in this review.

scholarly journals A structurally conserved RNA element within SARS-CoV-2 ORF1a RNA and S mRNA regulates translation in response to viral S protein-induced signaling in human lung cells.

2021 ◽  
Author(s):  
Abhijit Basu ◽  
Srinivasa Penumutchu ◽  
Kien Nguyen ◽  
Uri Mbonye ◽  
Blanton S. Tolbert ◽  
...  
Keyword(s):  
Host Cell ◽  
Translational Control ◽  
Viral Protein ◽  
Human Lung ◽  
Ribosomal Subunit ◽  
Lung Cells ◽  
Large Ribosomal Subunit ◽  
S Protein ◽  
Human Lung Cells ◽  
Rna Elements

The positive-sense, single-stranded RNA genome SARS-CoV-2 harbors functionally important cis-acting elements governing critical aspects of viral gene expression. However, insights on how these elements sense various signals from the host cell and regulate viral protein synthesis are lacking. Here, we identified two novel cis-regulatory elements in SARS-CoV-2 ORF1a and S RNAs and describe their role in translational control of SARS-CoV-2. These elements are sequence-unrelated but form conserved hairpin structures (validated by NMR) resembling G amma A ctivated I nhibitor of T ranslation (GAIT) elements that are found in a cohort of human mRNAs directing translational suppression in myeloid cells in response to IFN-γ. Our studies show that treatment of human lung cells with receptor-binding S1 subunit, S protein pseudotyped lentivirus, and S protein-containing virus-like particles triggers a signaling pathway involving DAP-kinase1 that leads to phosphorylation and release of the ribosomal protein L13a from the large ribosomal subunit. Released L13a forms a V irus A ctivated I nhibitor of T ranslation (VAIT) complex that binds to ORF1a and S VAIT elements, causing translational silencing. Translational silencing requires extracellular S protein (and its interaction with host ACE2 receptor), but not its intracellular synthesis. RNA-protein interaction analyses and in vitro translation experiments showed that GAIT and VAIT elements do not compete with each other, highlighting differences between the two pathways. Sequence alignments of SARS-CoV-2 genomes showed a high level of conservation of VAIT elements, suggesting their functional importance. This VAIT-mediated translational control mechanism of SARS-CoV-2 may provide novel targets for small molecule intervention and/or facilitate development of more effective mRNA vaccines. Importance Specific RNA elements in the genomes of RNA viruses play important roles in host-virus interaction. For SARS-CoV-2, the mechanistic insights on how these RNA elements could sense the signals from the host cell are lacking. Here we report a novel relationship between the GAIT-like SARS-CoV-2 RNA element (called VAITs) and the signal generated from the host cell. We show that for SARS-CoV-2, the interaction of spike protein with ACE2 not only serves the purpose for viral entry into the host cell, but also transduces signals that culminate into the phosphorylation and the release of L13a from the large ribosomal subunit. We also show that this event leads to the translational arrest of ORF1a and S mRNAs in a manner dependent on the structure of the RNA elements. Translational control of viral mRNA by a host-cell generated signal triggered by viral protein is a new paradigm in the host-virus relationship.

scholarly journals Distinct domains of Me31B interact with different eIF4E isoforms in the male germ line of Drosophila melanogaster

2021 ◽  
Author(s):  
Carla Layana ◽  
Emiliano S. Vilardo ◽  
Gonzalo Corujo ◽  
Greco Hernandez ◽  
Rolando Rivera-Pomar
Keyword(s):  
Drosophila Melanogaster ◽  
Translational Control ◽  
Germ Line ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Interacting Protein ◽  
Control Of Gene Expression ◽  
Amino Terminal ◽  
Key Factor

Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). In this study, we show that Drosophila eIF4E-1 and eIF4E-3 occur in PBs where might play a role in mRNA storage and translational repression. We also demonstrate that the DEAD-box RNA helicase Me31B, a component of PBs, physically interacts with eIF4E-1 and eIF4E-3 both in the yeast two-hybrid system and FRET in Drosophila S2 cells. Moreover, truncated and point mutated Me31B proteins indicate that the binding sites of Me31B for eIF4E-1 and eIF4E-3 are located in different domains. Residues Y401-L407 (at the carboxy-terminal) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminal) are critical for interaction with eIF4E-3. Thus, Me31B represents a novel type of eIF4E-interacting protein. Our observations suggest that Me31B might recognize different eIF4E isoforms in different tissues, which could be the key to silencing specific messengers. They provide further evidence that alternative forms of eIF4E and their interactions with various partners add complexity to the control of gene expression in eukaryotes.

scholarly journals Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

2019 ◽  
Author(s):  
Susan Wagner ◽  
Anna Herrmannová ◽  
Vladislava Hronová ◽  
Neelam Sen ◽  
Ross D. Hannan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Translational Control ◽  
Mammalian Cells ◽  
Regulation Of Gene Expression ◽  
Ribosome Profiling ◽  
Initiation Factor ◽  
Start Codon ◽  
Codon Recognition ◽  
Initiation Phase ◽  
Gene Expression Studies

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.

Nuclear localization of initiation factor 2 in neuron primary cultures

1995 ◽  
Vol 53 ◽  
pp. 774-775
Author(s):  
F.J. Martinez Alonso ◽  
M.V. Toledo Lobo ◽  
S. Rodriguez Martínez ◽  
F.M. Muñoz Postigo ◽  
J.J. López-Fando Castro
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Control Function ◽  
Primary Cultures ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
40S Ribosomal Subunit ◽  
Initiation Factor 2 ◽  
Immunocytochemical Techniques ◽  
Embryo Brain

The dominant mechanism that controls protein synthesis is the phosphorylation/dephosphorylation of initiation and elongation factors, with a translational control function. Each phase of protein synthesis is promoted by some of these factors that transiently interact with ribosomes, mRNAs and aminoacyltRNAs. Eukaryotic initiation factor-2 (eIF2, 130 kD) is one of these proteins and it is composed of three subunits: alpha, beta and gamma. eIF2 forms a ternary complex (GTP-eIF2-Met tRNAi) that can then interact with the 40S ribosomal subunit which in turn binds mRNA and the 60S ribosomal subunit to form the 80S initiation complex. The relation between eIF2 and the ribosomes is then a well established aspect of protein synthesis, but there are no previous studies about the distribution of eIF2 within the cell.Using immunocytochemical techniques, we show the distribution of eIF2 within the cell found in primary cultures of rat embryo brain neurons, in which eIF2 and eIF2-kinases have been identified. Primary culture neuron cells were grown in D15 and N2 mediums for 8 days.

scholarly journals Control of embryonic stem cell self-renewal and differentiation via coordinated alternative splicing and translation of YY2

2016 ◽  
Vol 113 (44) ◽  
pp. 12360-12367 ◽  
Author(s):  
Soroush Tahmasebi ◽  
Seyed Mehdi Jafarnejad ◽  
Ingrid S. Tam ◽  
Thomas Gonatopoulos-Pournatzis ◽  
Edna Matta-Camacho ◽  
...  
Keyword(s):  
Translational Control ◽  
Binding Protein ◽  
Critical Role ◽  
Embryonic Stem ◽  
Initiation Factor ◽  
Self Renewal ◽  
Control Of Gene Expression ◽  
Eukaryotic Initiation Factor 4E ◽  
Polypyrimidine Tract Binding Protein ◽  
Splicing Regulator

Translational control of gene expression plays a key role during the early phases of embryonic development. Here we describe a transcriptional regulator of mouse embryonic stem cells (mESCs), Yin-yang 2 (YY2), that is controlled by the translation inhibitors, Eukaryotic initiation factor 4E-binding proteins (4E-BPs). YY2 plays a critical role in regulating mESC functions through control of key pluripotency factors, including Octamer-binding protein 4 (Oct4) and Estrogen-related receptor-β (Esrrb). Importantly, overexpression of YY2 directs the differentiation of mESCs into cardiovascular lineages. We show that the splicing regulator Polypyrimidine tract-binding protein 1 (PTBP1) promotes the retention of an intron in the 5′-UTR of Yy2 mRNA that confers sensitivity to 4E-BP–mediated translational suppression. Thus, we conclude that YY2 is a major regulator of mESC self-renewal and lineage commitment and document a multilayer regulatory mechanism that controls its expression.

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