scholarly journals Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons

2017 ◽  
Vol 114 (11) ◽  
pp. E2126-E2135 ◽  
Author(s):  
Jinsheng Dong ◽  
Colin Echeverría Aitken ◽  
Anil Thakur ◽  
Byung-Sik Shin ◽  
Jon R. Lorsch ◽  
...  
Keyword(s):  
Initiation Factor ◽  
Start Codon ◽  
Entry And Exit ◽  
Functional Importance ◽  
Conserved Residues ◽  
Kozak Context ◽  
Domains Of Life ◽  
Mrna Binding ◽  
Yeast Mutants

The eukaryotic 43S preinitiation complex (PIC) bearing Met-tRNAiMet in a ternary complex (TC) with eukaryotic initiation factor (eIF)2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition provokes rearrangement from an open PIC conformation with TC bound in a state not fully engaged with the P site (“POUT”) to a closed, arrested conformation with TC tightly bound in the “PIN” state. Yeast ribosomal protein Rps3/uS3 resides in the mRNA entry channel of the 40S subunit and contacts mRNA via conserved residues whose functional importance was unknown. We show that substitutions of these residues reduce bulk translation initiation and diminish initiation at near-cognate UUG start codons in yeast mutants in which UUG selection is abnormally high. Two such substitutions—R116D and R117D—also increase discrimination against an AUG codon in suboptimal Kozak context. Consistently, the Arg116 and Arg117 substitutions destabilize TC binding to 48S PICs reconstituted in vitro with mRNA harboring a UUG start codon, indicating destabilization of the closed PIN state with a UUG–anticodon mismatch. Using model mRNAs lacking contacts with either the mRNA entry or exit channels of the 40S subunit, we demonstrate that Arg116/Arg117 are crucial for stabilizing PIC–mRNA contacts at the entry channel, augmenting the function of eIF3 at both entry and exit channels. The corresponding residues in bacterial uS3 promote the helicase activity of the elongating ribosome, suggesting that uS3 contacts with mRNA enhance multiple phases of translation across different domains of life.

scholarly journals Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons

2017 ◽  
Author(s):  
Jinsheng Dong ◽  
Colin Echeverría Aitken ◽  
Anil Thakur ◽  
Byung-Sik Shin ◽  
Jon R. Lorsch ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Start Codon ◽  
Entry And Exit ◽  
Functional Importance ◽  
Conserved Residues ◽  
Kozak Context ◽  
Domains Of Life ◽  
Mrna Binding ◽  
Yeast Mutants

ABSTRACTThe eukaryotic 43S pre-initiation complex (PIC) bearing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition provokes rearrangement from an open PIC conformation with TC bound in a state not fully engaged with the P site (“POUT”) to a closed, arrested conformation with TC tightly bound in the “PIN” state. Yeast ribosomal protein Rps3/uS3 resides in the mRNA entry channel of the 40S subunit and contacts mRNA via conserved residues whose functional importance was unknown. We show that substitutions of these residues reduce bulk translation initiation and diminish initiation at near-cognate UUG start codons in yeast mutants in which UUG selection is abnormally high (Sui-), conferring the Ssu- phenotype. Two such Ssu- substitutions—R116D and R117D—also increase discrimination against an AUG codon in suboptimal Kozak context. Consistently, the Arg116 and Arg117 substitutions destabilize TC binding to 48S PICs reconstituted in vitro with mRNA harboring a UUG start codon, indicating destabilization of the closed PIN state with a UUG:anticodon mismatch. Using model mRNAs lacking contacts with either the mRNA entry or exit channels of the 40S subunit, we demonstrate that Arg116/Arg117 are crucial for stabilizing PIC:mRNA contacts at the entry channel, complementing the function of eIF3 at both entry and exit channels. The corresponding residues in bacterial uS3 promote the helicase activity of the elongating ribosome, suggesting that uS3 contacts with mRNA enhance multiple phases of translation across different domains of life.

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 Identification and Characterization of Functionally Critical, Conserved Motifs in the Internal Repeats and N-terminal Domain of Yeast Translation Initiation Factor 4B (yeIF4B)

2013 ◽  
Vol 289 (3) ◽  
pp. 1704-1722 ◽  
Author(s):  
Fujun Zhou ◽  
Sarah E. Walker ◽  
Sarah F. Mitchell ◽  
Jon R. Lorsch ◽  
Alan G. Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Start Codon ◽  
Rna Recognition Motif ◽  
Conserved Motifs ◽  
Terminal Domain ◽  
Sequence Elements

eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC.

scholarly journals Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts with eIF5

1998 ◽  
Vol 18 (8) ◽  
pp. 4935-4946 ◽  
Author(s):  
Lon Phan ◽  
Xiaolong Zhang ◽  
Katsura Asano ◽  
James Anderson ◽  
Hans-Peter Vornlocher ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Gel Filtration ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Start Codon ◽  
Binding Assays ◽  
Core Complex ◽  
Eukaryotic Translation ◽  
Translation Initiation Factor 3

ABSTRACT Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in theSaccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of ≈600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAi Met binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.

scholarly journals Functional Cyclization of Eukaryotic mRNAs

2020 ◽  
Vol 21 (5) ◽  
pp. 1677 ◽  
Author(s):  
Olga Alekhina ◽  
Ilya Terenin ◽  
Sergey Dmitriev ◽  
Konstantin Vassilenko
Keyword(s):  
Closed Loop ◽  
Mrna Translation ◽  
Inhibitory Effect ◽  
Dominant Negative ◽  
In Situ Monitoring ◽  
Initiation Factor ◽  
Start Codon ◽  
Loop Model ◽  
Mrna Molecule

The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions that brings 5′ and 3′ ends together. This circularization is thought to promote the engagement of terminating ribosomes to a new round of translation at the same mRNA molecule, thus enhancing protein synthesis. Despite the general acceptance and the elegance of the hypothesis, it has never been proved experimentally. Using continuous in situ monitoring of luciferase synthesis in a mammalian in vitro system, we show here that the rate of translation initiation at capped and polyadenylated reporter mRNAs increases after the time required for the first ribosomes to complete mRNA translation. Such acceleration strictly requires the presence of a poly(A)-tail and is abrogated by the addition of poly(A) RNA fragments or m7GpppG cap analog to the translation reaction. The optimal functional interaction of mRNA termini requires 5′ untranslated region (UTR) and 3′ UTR of moderate lengths and provides stronger acceleration, thus a longer poly(A)-tail. Besides, we revealed that the inhibitory effect of the dominant negative R362Q mutant of initiation factor eIF4A diminishes in the course of translation reaction, suggesting a relaxed requirement for ATP. Taken together, our results imply that, upon the functional looping of an mRNA, the recycled ribosomes can be recruited to the start codon of the same mRNA molecule in an eIF4A-independent fashion. This non-canonical closed-loop assisted reinitiation (CLAR) mode provides efficient translation of the functionally circularized mRNAs.

scholarly journals The β Subunit of Eukaryotic Translation Initiation Factor 2 Binds mRNA through the Lysine Repeats and a Region Comprising the C2-C2 Motif

1999 ◽  
Vol 19 (1) ◽  
pp. 173-181 ◽  
Author(s):  
Jomar P. Laurino ◽  
Glória M. Thompson ◽  
Eliza Pacheco ◽  
Beatriz A. Castilho
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Initiation Factor 2 ◽  
Lysine Residues ◽  
Eukaryotic Translation Initiation ◽  
Mrna Binding ◽  
Translation Initiation Factor 2

ABSTRACT Eukaryotic translation initiation factor 2 (eIF2) has been implicated in the selection of the AUG codon as the start site for eukaryotic translation initiation, since mutations in its three subunits in yeast that allow the recognition of a UUG codon by the anticodon of the initiator Met-tRNAMet have been identified. All such mutations in the beta subunit of eIF2 (eIF2β) mapped to a region containing a putative zinc finger structure of the C2-C2 type, indicating that these sequences could be involved in RNA recognition. Another feature of eIF2β that could mediate an interaction with RNA is located in the amino-terminal sequences and is composed of three repeats of seven lysine residues which are highly conserved in other species. We show here the ability of eIF2β, purified from Escherichia coli as a fusion to glutathione S-transferase, to bind mRNA in vitro. Through a deletion analysis, mRNA binding was found to be dependent on the lysine repeats and a region encompassing the C2-C2motif. Strong mRNA binding in vitro could be maintained by the presence of only one lysine or one arginine run but not one alanine run. We further show that only one run of lysine residues is sufficient for the in vivo function of eIF2β, probably through charge interaction, since its replacement by arginines did not impair cell viability, whereas substitution for alanines resulted in inviable cells. mRNA binding, but not GTP-dependent initiator Met-tRNAMet binding, by the eIF2 complex was determined to be dependent on the presence of the lysine runs of the beta subunit.

scholarly journals Eukaryotic Translation Initiation Factor 3 (eIF3) and eIF2 Can Promote mRNA Binding to 40S Subunits Independently of eIF4G in Yeast

2006 ◽  
Vol 26 (4) ◽  
pp. 1355-1372 ◽  
Author(s):  
Antonina V. Jivotovskaya ◽  
Leoš Valášek ◽  
Alan G. Hinnebusch ◽  
Klaus H. Nielsen
Keyword(s):  
Ternary Complex ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Initiation Factors ◽  
Eukaryotic Translation Initiation ◽  
Mrna Binding ◽  
Rate Limiting

ABSTRACT Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAi Met 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.

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

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jose Luis Llácer ◽  
Tanweer Hussain ◽  
Adesh K Saini ◽  
Jagpreet Singh Nanda ◽  
Sukhvir Kaur ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Start Codon ◽  
Dual Function ◽  
Translational Initiation ◽  
Eukaryotic Translation ◽  
Codon Recognition ◽  
40S Ribosomal Subunit ◽  
Eukaryotic Translation Initiation

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.

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.

Discovery of N-Phenyl-4-(1H-pyrrol-3-yl)pyrimidin-2-amine Derivatives as Potent Mnk2 Inhibitors: Design, Synthesis, SAR Analysis, and Evaluation of in vitro Anti-leukaemic Activity

2019 ◽  
Vol 15 (6) ◽  
pp. 602-623 ◽  
Author(s):  
Ahmed M. Abdelaziz ◽  
Sarah Diab ◽  
Saiful Islam ◽  
Sunita K.C. Basnet ◽  
Benjamin Noll ◽  
...  
Keyword(s):  
Proliferative Activity ◽  
Myeloid Cell ◽  
Structural Diversity ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cell Leukaemia ◽  
Aberrant Expression ◽  
Design Synthesis ◽  
Induced Apoptosis

Background:Aberrant expression of eukaryotic translation initiation factor 4E (eIF4E) is common in many types of cancer including acute myeloid leukaemia (AML). Phosphorylation of eIF4E by MAPK-interacting kinases (Mnks) is essential for the eIF4E-mediated oncogenic activity. As such, the pharmacological inhibition of Mnks can be an effective strategy for the treatment of cancer.Methods:A series of N-phenyl-4-(1H-pyrrol-3-yl)pyrimidin-2-amine derivatives was designed and synthesised. The Mnk inhibitory activity of these derivatives as well as their anti-proliferative activity against MV4-11 AML cells was determined.Results:These compounds were identified as potent Mnk2 inhibitors. Most of them demonstrated potent anti-proliferative activity against MV4-11 AML cells. The cellular mechanistic studies of the representative inhibitors revealed that they reduced the level of phosphorylated eIF4E and induced apoptosis by down-regulating the anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1) and by cleaving poly(ADP-ribose)polymerase (PARP). The lead compound 7k possessed desirable pharmacokinetic properties and oral bioavailability.Conclusion:This work proposes that exploration of the structural diversity in the context of Nphenyl- 4-(1H-pyrrol-3-yl)pyrimidin-2-amine would offer potent and selective Mnk inhibitors.

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