competent e coli cells  (Thermo Fisher)


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    Name:
    Competent Cell Sampler
    Description:
    Our Competent Cell Sampler includes two reactions of four chemically competent E coli strains One Shot Mach1 T1R catalog C862003 is the fastest growing chemically competent strain currently available Mach1 T1R colonies are clearly visible within eight hours of plating your transformation From an overnight colony minipreps can be performed after four hours of growth Genotype F Φ80lacZΔM15 ΔlacX74 hsdR rK mK ΔrecA1398 endA1 tonA One Shot OmniMAX 2 T1R cells catalog C854003 are perfect for use in all cloning applications including Gateway technology They offer the highest transformation efficiency of any chemically competent cell in the One Shot packaging format Genotype F proAB lacIq lacZΔM15 Tn10 TetR Δ ccdAB mcrA Δ mrr hsdRMS mcrBC Φ80lacZΔM15 Δ lacZYA argF U169 endA1 recA1 supE44 thi 1 gyrA96 relA1 tonA panD One Shot Stbl3 catalog C7373 03 reduce the frequency of unwanted homologous recombination and so are perfect for cloning unstable DNA Genotype F mcrB mrr hsdS20 rB mB recA13 supE44 ara 14 galK2 lacY1 proA2 rpsL20 StrR xyl 5 leu mtl 1 One Shot TOP10 cells C4040 03 are perfect for routine cloning and included in many TOPO cloning and expression kits Genotype F mcrA Δ mrr hsdRMS mcrBC Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ ara leu 7697 galU galK rpsL StrR endA1 nupGTry them all to optimize transformations for your favorite clones
    Catalog Number:
    a10469
    Price:
    None
    Applications:
    Chemically Competent Cells for Cloning|Cloning|Transformation
    Category:
    Competent Cells Strains
    Buy from Supplier


    Structured Review

    Thermo Fisher competent e coli cells
    Our Competent Cell Sampler includes two reactions of four chemically competent E coli strains One Shot Mach1 T1R catalog C862003 is the fastest growing chemically competent strain currently available Mach1 T1R colonies are clearly visible within eight hours of plating your transformation From an overnight colony minipreps can be performed after four hours of growth Genotype F Φ80lacZΔM15 ΔlacX74 hsdR rK mK ΔrecA1398 endA1 tonA One Shot OmniMAX 2 T1R cells catalog C854003 are perfect for use in all cloning applications including Gateway technology They offer the highest transformation efficiency of any chemically competent cell in the One Shot packaging format Genotype F proAB lacIq lacZΔM15 Tn10 TetR Δ ccdAB mcrA Δ mrr hsdRMS mcrBC Φ80lacZΔM15 Δ lacZYA argF U169 endA1 recA1 supE44 thi 1 gyrA96 relA1 tonA panD One Shot Stbl3 catalog C7373 03 reduce the frequency of unwanted homologous recombination and so are perfect for cloning unstable DNA Genotype F mcrB mrr hsdS20 rB mB recA13 supE44 ara 14 galK2 lacY1 proA2 rpsL20 StrR xyl 5 leu mtl 1 One Shot TOP10 cells C4040 03 are perfect for routine cloning and included in many TOPO cloning and expression kits Genotype F mcrA Δ mrr hsdRMS mcrBC Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ ara leu 7697 galU galK rpsL StrR endA1 nupGTry them all to optimize transformations for your favorite clones
    https://www.bioz.com/result/competent e coli cells/product/Thermo Fisher
    Average 99 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    competent e coli cells - by Bioz Stars, 2020-11
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: The olfactory secretome varies according to season in female sheep and goat
    Article Snippet: .. PCR products were cloned into pCR4®-TOPO vector (TOPO™-TA cloning™ kit, Invitrogen), then amplified into Escherichia coli One Shot™ Top10 chemically competent cells (Invitrogen). .. Recombinant plasmids were purified with QIAprep Spin Miniprep kit (Qiagen) and sequenced in both senses (Eurofins Genomics).

    Amplification:

    Article Title: The olfactory secretome varies according to season in female sheep and goat
    Article Snippet: .. PCR products were cloned into pCR4®-TOPO vector (TOPO™-TA cloning™ kit, Invitrogen), then amplified into Escherichia coli One Shot™ Top10 chemically competent cells (Invitrogen). .. Recombinant plasmids were purified with QIAprep Spin Miniprep kit (Qiagen) and sequenced in both senses (Eurofins Genomics).

    Ligation:

    Article Title: Identification of an anti-idiotypic antibody that defines a B-cell subset(s) producing xenoantibodies in primates
    Article Snippet: .. – Purified PCR products were ligated into a pCR®2·1 vector (TA Cloning Kit; Invitrogen, Carlsbad, CA) and INVαF′ One Shot™ competent cells (Invitrogen) were transformed with the ligation reactions and plated onto Xgal-containing Luria Broth (LB) ampicillin plates. .. The DNA sequence was analysed for a total of 100 cDNA clones that were prepared from the VH 3 cDNA libraries.

    TA Cloning:

    Article Title: Identification of an anti-idiotypic antibody that defines a B-cell subset(s) producing xenoantibodies in primates
    Article Snippet: .. – Purified PCR products were ligated into a pCR®2·1 vector (TA Cloning Kit; Invitrogen, Carlsbad, CA) and INVαF′ One Shot™ competent cells (Invitrogen) were transformed with the ligation reactions and plated onto Xgal-containing Luria Broth (LB) ampicillin plates. .. The DNA sequence was analysed for a total of 100 cDNA clones that were prepared from the VH 3 cDNA libraries.

    Construct:

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol
    Article Snippet: .. This construct was transformed into TOP10 competent cells (Invitrogen) for expression. ..

    Purification:

    Article Title: Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells
    Article Snippet: .. After purification of the plasmids from the transformed Top10 competent cells (Invitrogen), the sequences of the cDNA inserts were verified with a Taq cycle sequencing kit (Takara Biomedicals) and an autofluorimetric DNA sequencer (310 Genetic Analyzer; Applied Biosystems). .. The cDNA inserts were then transferred to the pAd/CMV/V5-DEST vector (Invitrogen) by means of the Gateway system using LR clonase (Invitrogen).

    Article Title: Identification of an anti-idiotypic antibody that defines a B-cell subset(s) producing xenoantibodies in primates
    Article Snippet: .. – Purified PCR products were ligated into a pCR®2·1 vector (TA Cloning Kit; Invitrogen, Carlsbad, CA) and INVαF′ One Shot™ competent cells (Invitrogen) were transformed with the ligation reactions and plated onto Xgal-containing Luria Broth (LB) ampicillin plates. .. The DNA sequence was analysed for a total of 100 cDNA clones that were prepared from the VH 3 cDNA libraries.

    Article Title: Laminin N-terminus α31 expression during development in an inducible-transgenic mouse model is lethal and causes a multitude of tissue-specific defects
    Article Snippet: .. Purified inserts were ligated into vectors at 3:1 molar ratios, either using Instant Sticky-end Ligase Master Mix (New England Biolabs) following manufacturers protocol, or using 400 U of T4 DNA ligase and 1X reaction buffer (50 mM Tris-HCl, 10 mM MgCl2 1 mM ATP, 10 mM DTT, New England Biolabs) at 16°C overnight, followed by enzymatic inactivation at 65°C for 10 min. Ligated DNA was heat-shock transformed into One-Shot TOP10 chemically competent E. coli cells (Thermo Fisher Scientific) following manufacturer’s protocol, then plated onto LB plates containing the appropriate antibiotic (100 μg/ml ampicillin, 50 μg/ml kanamycin or 25 μg/ml chloramphenicol, Sigma Aldrich). .. Plasmid DNA was extracted from bacteria using GenElute™ Plasmid Miniprep Kit (Sigma Aldrich), following the manufacturer’s protocol.

    Sequencing:

    Article Title: Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells
    Article Snippet: .. After purification of the plasmids from the transformed Top10 competent cells (Invitrogen), the sequences of the cDNA inserts were verified with a Taq cycle sequencing kit (Takara Biomedicals) and an autofluorimetric DNA sequencer (310 Genetic Analyzer; Applied Biosystems). .. The cDNA inserts were then transferred to the pAd/CMV/V5-DEST vector (Invitrogen) by means of the Gateway system using LR clonase (Invitrogen).

    Incubation:

    Article Title: What Goes in Must Come out: Testing for Biases in Molecular Analysis of Arbuscular Mycorrhizal Fungal Communities
    Article Snippet: .. Vectors (4µl) were transformed into Escherichia coli cells by incubation with 25µl competent E. coli cells (DH5α; Invitrogen, Renfrewshire, UK) at 4°C for 30 minutes, followed by a heatshock of 42°C for 45 seconds and rotary incubation with 475µl Super Optimal broth with Catabolite repression (SOC) at 37°C for 1 hour. ..

    Expressing:

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol
    Article Snippet: .. This construct was transformed into TOP10 competent cells (Invitrogen) for expression. ..

    Polymerase Chain Reaction:

    Article Title: Identification of an anti-idiotypic antibody that defines a B-cell subset(s) producing xenoantibodies in primates
    Article Snippet: .. – Purified PCR products were ligated into a pCR®2·1 vector (TA Cloning Kit; Invitrogen, Carlsbad, CA) and INVαF′ One Shot™ competent cells (Invitrogen) were transformed with the ligation reactions and plated onto Xgal-containing Luria Broth (LB) ampicillin plates. .. The DNA sequence was analysed for a total of 100 cDNA clones that were prepared from the VH 3 cDNA libraries.

    Article Title: The olfactory secretome varies according to season in female sheep and goat
    Article Snippet: .. PCR products were cloned into pCR4®-TOPO vector (TOPO™-TA cloning™ kit, Invitrogen), then amplified into Escherichia coli One Shot™ Top10 chemically competent cells (Invitrogen). .. Recombinant plasmids were purified with QIAprep Spin Miniprep kit (Qiagen) and sequenced in both senses (Eurofins Genomics).

    Transformation Assay:

    Article Title: Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells
    Article Snippet: .. After purification of the plasmids from the transformed Top10 competent cells (Invitrogen), the sequences of the cDNA inserts were verified with a Taq cycle sequencing kit (Takara Biomedicals) and an autofluorimetric DNA sequencer (310 Genetic Analyzer; Applied Biosystems). .. The cDNA inserts were then transferred to the pAd/CMV/V5-DEST vector (Invitrogen) by means of the Gateway system using LR clonase (Invitrogen).

    Article Title: Identification of an anti-idiotypic antibody that defines a B-cell subset(s) producing xenoantibodies in primates
    Article Snippet: .. – Purified PCR products were ligated into a pCR®2·1 vector (TA Cloning Kit; Invitrogen, Carlsbad, CA) and INVαF′ One Shot™ competent cells (Invitrogen) were transformed with the ligation reactions and plated onto Xgal-containing Luria Broth (LB) ampicillin plates. .. The DNA sequence was analysed for a total of 100 cDNA clones that were prepared from the VH 3 cDNA libraries.

    Article Title: What Goes in Must Come out: Testing for Biases in Molecular Analysis of Arbuscular Mycorrhizal Fungal Communities
    Article Snippet: .. Vectors (4µl) were transformed into Escherichia coli cells by incubation with 25µl competent E. coli cells (DH5α; Invitrogen, Renfrewshire, UK) at 4°C for 30 minutes, followed by a heatshock of 42°C for 45 seconds and rotary incubation with 475µl Super Optimal broth with Catabolite repression (SOC) at 37°C for 1 hour. ..

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol
    Article Snippet: .. This construct was transformed into TOP10 competent cells (Invitrogen) for expression. ..

    Article Title: Laminin N-terminus α31 expression during development in an inducible-transgenic mouse model is lethal and causes a multitude of tissue-specific defects
    Article Snippet: .. Purified inserts were ligated into vectors at 3:1 molar ratios, either using Instant Sticky-end Ligase Master Mix (New England Biolabs) following manufacturers protocol, or using 400 U of T4 DNA ligase and 1X reaction buffer (50 mM Tris-HCl, 10 mM MgCl2 1 mM ATP, 10 mM DTT, New England Biolabs) at 16°C overnight, followed by enzymatic inactivation at 65°C for 10 min. Ligated DNA was heat-shock transformed into One-Shot TOP10 chemically competent E. coli cells (Thermo Fisher Scientific) following manufacturer’s protocol, then plated onto LB plates containing the appropriate antibiotic (100 μg/ml ampicillin, 50 μg/ml kanamycin or 25 μg/ml chloramphenicol, Sigma Aldrich). .. Plasmid DNA was extracted from bacteria using GenElute™ Plasmid Miniprep Kit (Sigma Aldrich), following the manufacturer’s protocol.

    Plasmid Preparation:

    Article Title: Identification of an anti-idiotypic antibody that defines a B-cell subset(s) producing xenoantibodies in primates
    Article Snippet: .. – Purified PCR products were ligated into a pCR®2·1 vector (TA Cloning Kit; Invitrogen, Carlsbad, CA) and INVαF′ One Shot™ competent cells (Invitrogen) were transformed with the ligation reactions and plated onto Xgal-containing Luria Broth (LB) ampicillin plates. .. The DNA sequence was analysed for a total of 100 cDNA clones that were prepared from the VH 3 cDNA libraries.

    Article Title: The olfactory secretome varies according to season in female sheep and goat
    Article Snippet: .. PCR products were cloned into pCR4®-TOPO vector (TOPO™-TA cloning™ kit, Invitrogen), then amplified into Escherichia coli One Shot™ Top10 chemically competent cells (Invitrogen). .. Recombinant plasmids were purified with QIAprep Spin Miniprep kit (Qiagen) and sequenced in both senses (Eurofins Genomics).

    Article Title: Evaluation of a Campylobacter fetus subspecies venerealis real-time quantitative polymerase chain reaction for direct analysis of bovine preputial samples
    Article Snippet: .. The 142 bp product was ligated into the pGEM-T Easy plasmid and used to transform competent E. coli JM109 cells (Invitrogen, Burlington, Ontario). .. Plasmids with the target insert (termed par A-containing plasmids) were confirmed by sequencing, quantified, and used as qPCR assay standards.

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  • 90
    Thermo Fisher e coli cells expressing eif4a wt
    RocA clamps <t>eIF4A</t> on polypurine motif even after ATP hydrolysis (a, b) Direct measurement of the eIF4A/RNA affinity by fluorescence polarization for eIF4A and 5′ FAM-labeled RNAs in the presence or absence of RocA. Data represent mean and S.D. (n = 3). (c) Motif enrichments along entire 4-mer motifs in Bind-n-Seq with ADP + Pi and highest-scoring elements (inset). (d) Competition assay with unlabeled RNA. Data represent mean (n = 3). (e) Ribosome toeprinting assay performed in RRL in the presence of GMP-PNP in the presence or absence of 3 μM RocA treatment. (f) Relative RNase I cleavage protected by eIF4A/RocA complex on mRNA containg one AGAGAG at the middle in footprinting assay. See the original data in Extended Data Figure 9f .
    E Coli Cells Expressing Eif4a Wt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli cells expressing eif4a wt/product/Thermo Fisher
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e coli cells expressing eif4a wt - by Bioz Stars, 2020-11
    90/100 stars
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    RocA clamps eIF4A on polypurine motif even after ATP hydrolysis (a, b) Direct measurement of the eIF4A/RNA affinity by fluorescence polarization for eIF4A and 5′ FAM-labeled RNAs in the presence or absence of RocA. Data represent mean and S.D. (n = 3). (c) Motif enrichments along entire 4-mer motifs in Bind-n-Seq with ADP + Pi and highest-scoring elements (inset). (d) Competition assay with unlabeled RNA. Data represent mean (n = 3). (e) Ribosome toeprinting assay performed in RRL in the presence of GMP-PNP in the presence or absence of 3 μM RocA treatment. (f) Relative RNase I cleavage protected by eIF4A/RocA complex on mRNA containg one AGAGAG at the middle in footprinting assay. See the original data in Extended Data Figure 9f .

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: RocA clamps eIF4A on polypurine motif even after ATP hydrolysis (a, b) Direct measurement of the eIF4A/RNA affinity by fluorescence polarization for eIF4A and 5′ FAM-labeled RNAs in the presence or absence of RocA. Data represent mean and S.D. (n = 3). (c) Motif enrichments along entire 4-mer motifs in Bind-n-Seq with ADP + Pi and highest-scoring elements (inset). (d) Competition assay with unlabeled RNA. Data represent mean (n = 3). (e) Ribosome toeprinting assay performed in RRL in the presence of GMP-PNP in the presence or absence of 3 μM RocA treatment. (f) Relative RNase I cleavage protected by eIF4A/RocA complex on mRNA containg one AGAGAG at the middle in footprinting assay. See the original data in Extended Data Figure 9f .

    Article Snippet: Pulldown assay The lysate of E. coli cells expressing eIF4A WT or eIF4A D296A-T298K proteins from 1 ml culture was prepared as described in “Purification of recombinant eIF4A proteins“ and incubated with 10 μl of HisPur Ni-NTA Magnetic Beads (Thermo Scientific) at 4 °C for 30 min.

    Techniques: Fluorescence, Labeling, Competitive Binding Assay, Toeprinting Assay, Footprinting

    Purification of SBP-tagged eIF4A and co-purified RNA from HEK 293 cells (a) Western blot of exogenous SBP-eIF4A and endogenous eIF4A in tetracycline-inducible stable cell line. Expression of physiological levels of the tagged allele attenuated endogenous eIF4A expression but preserved overall eIF4A levels, likely reflecting the same feedback loop previously reported between eIF4AI and eIF4AII 31 . (b) CBB staining of purified SBP-eIF4A and SYBR Gold staining of purified RNA bound to SBP-eIF4A with or without Micrococcal Nuclease (MNase). (c) Correlation of sum of the mRNA fragment reads of each transcript between biological replicates of RIP-seq. r is Pearson’s correlation coefficient. P value is calculated by Student′s t-test. (d) Histogram of the number of transcripts along RNA/eIF4A interaction -fold change by RIP-Seq when cells are treated with 0.03 or 0.3 µM RocA normalized to spiked-in RNA. Data present the same mRNAs analyzed in Figure 1a . Median -fold change is shown. Bin width is 0.1. (e) Correlation of RIP -fold change between different concentration of RocA treatments. ρ: Spearman’s rank correlation coefficient. (f) Correlation of translation -fold change to RIP -fold change with the same concentration of RocA treatment. ρ: Spearman’s rank correlation.

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: Purification of SBP-tagged eIF4A and co-purified RNA from HEK 293 cells (a) Western blot of exogenous SBP-eIF4A and endogenous eIF4A in tetracycline-inducible stable cell line. Expression of physiological levels of the tagged allele attenuated endogenous eIF4A expression but preserved overall eIF4A levels, likely reflecting the same feedback loop previously reported between eIF4AI and eIF4AII 31 . (b) CBB staining of purified SBP-eIF4A and SYBR Gold staining of purified RNA bound to SBP-eIF4A with or without Micrococcal Nuclease (MNase). (c) Correlation of sum of the mRNA fragment reads of each transcript between biological replicates of RIP-seq. r is Pearson’s correlation coefficient. P value is calculated by Student′s t-test. (d) Histogram of the number of transcripts along RNA/eIF4A interaction -fold change by RIP-Seq when cells are treated with 0.03 or 0.3 µM RocA normalized to spiked-in RNA. Data present the same mRNAs analyzed in Figure 1a . Median -fold change is shown. Bin width is 0.1. (e) Correlation of RIP -fold change between different concentration of RocA treatments. ρ: Spearman’s rank correlation coefficient. (f) Correlation of translation -fold change to RIP -fold change with the same concentration of RocA treatment. ρ: Spearman’s rank correlation.

    Article Snippet: Pulldown assay The lysate of E. coli cells expressing eIF4A WT or eIF4A D296A-T298K proteins from 1 ml culture was prepared as described in “Purification of recombinant eIF4A proteins“ and incubated with 10 μl of HisPur Ni-NTA Magnetic Beads (Thermo Scientific) at 4 °C for 30 min.

    Techniques: Purification, Western Blot, Stable Transfection, Expressing, Staining, Concentration Assay

    eIF4A/RNA affinity measured by fluorescence polarization (a) CBB staining of recombinant proteins used in this study. (b) Summary of K d between RNA and eIF4A among the conditions assayed. (c, e-g, i) Direct measurement of the eIF4A/RNA affinity by fluorescence polarization for eIF4A WT, eIF4A (VX 4 GKT), or eIF4A (D296A-T298K) and 5′ FAM-labeled RNAs in the presence or absence of RocA. Data represent mean and S.D. (n = 3). (d) ATP crosslinking assay with eIF4A WT and eIF4A (VX 4 GKT). (h) Pulldown assay with His-MBP-eIF4A expressed in E. coli and eIF4E/G in RRL.

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: eIF4A/RNA affinity measured by fluorescence polarization (a) CBB staining of recombinant proteins used in this study. (b) Summary of K d between RNA and eIF4A among the conditions assayed. (c, e-g, i) Direct measurement of the eIF4A/RNA affinity by fluorescence polarization for eIF4A WT, eIF4A (VX 4 GKT), or eIF4A (D296A-T298K) and 5′ FAM-labeled RNAs in the presence or absence of RocA. Data represent mean and S.D. (n = 3). (d) ATP crosslinking assay with eIF4A WT and eIF4A (VX 4 GKT). (h) Pulldown assay with His-MBP-eIF4A expressed in E. coli and eIF4E/G in RRL.

    Article Snippet: Pulldown assay The lysate of E. coli cells expressing eIF4A WT or eIF4A D296A-T298K proteins from 1 ml culture was prepared as described in “Purification of recombinant eIF4A proteins“ and incubated with 10 μl of HisPur Ni-NTA Magnetic Beads (Thermo Scientific) at 4 °C for 30 min.

    Techniques: Fluorescence, Staining, Recombinant, Labeling

    RNA Bind-n-Seq and iCLIP reveal that RocA preferentially increases the affinity between eIF4A and polypurine motif (a) Correlations between 4-mer motif enrichment in Bind-n-Seq by 0.03 µM RocA treatment and motif prediction of 0.03 µM RocA effect in RIP-Seq. ρ: Spearman’s rank correlation. (b) Highest-scoring elements in Bind-n-Seq and RIP-Seq. (c) The change in mRNA binding for mRNAs with or without the enriched 4-mer motif (b) in their 5′ UTRs is shown as the RIP -fold change by RocA normalized to spike-in RNA. Significance is calculated by Mann-Whitney U test. (d) Enrichment of 4-mer motifs (b) in iCLIP by RocA treatment relative to control DMSO treatment. (e) The frequency of the 4-mer motif (b) in the 5′ UTR predicts whether a mRNA is high- or low-sensitivity, based on the difference in cumulative distributions of motifs in the 5′ UTR. Significance is calculated by Mann-Whitney U test. (f) Reporter assay in HEK 293 cells with a CAA-repeat 5′ UTR containing seven polypurine motif (AGAGAG) insertions ( Extended Data Figure 9a ). Data represent mean and S.D. (n = 3).

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: RNA Bind-n-Seq and iCLIP reveal that RocA preferentially increases the affinity between eIF4A and polypurine motif (a) Correlations between 4-mer motif enrichment in Bind-n-Seq by 0.03 µM RocA treatment and motif prediction of 0.03 µM RocA effect in RIP-Seq. ρ: Spearman’s rank correlation. (b) Highest-scoring elements in Bind-n-Seq and RIP-Seq. (c) The change in mRNA binding for mRNAs with or without the enriched 4-mer motif (b) in their 5′ UTRs is shown as the RIP -fold change by RocA normalized to spike-in RNA. Significance is calculated by Mann-Whitney U test. (d) Enrichment of 4-mer motifs (b) in iCLIP by RocA treatment relative to control DMSO treatment. (e) The frequency of the 4-mer motif (b) in the 5′ UTR predicts whether a mRNA is high- or low-sensitivity, based on the difference in cumulative distributions of motifs in the 5′ UTR. Significance is calculated by Mann-Whitney U test. (f) Reporter assay in HEK 293 cells with a CAA-repeat 5′ UTR containing seven polypurine motif (AGAGAG) insertions ( Extended Data Figure 9a ). Data represent mean and S.D. (n = 3).

    Article Snippet: Pulldown assay The lysate of E. coli cells expressing eIF4A WT or eIF4A D296A-T298K proteins from 1 ml culture was prepared as described in “Purification of recombinant eIF4A proteins“ and incubated with 10 μl of HisPur Ni-NTA Magnetic Beads (Thermo Scientific) at 4 °C for 30 min.

    Techniques: Binding Assay, MANN-WHITNEY, Reporter Assay, Cellular Antioxidant Activity Assay

    Characterization of toeprinting assay (a) Diagram of the reporters used in this study. (b, and c) In vitro translation in RRL with mRNAs containing seven polypurine motif (AGAGAG) insertions (b) and qPCR from the samples (c). (d) Dideoxy terminated sequencing of RNA by reverse transcription verified the toeprinting product length terminated by 48S ribosomes. (e) Ribosome toeprinting assay performed in RRL in the presence of m7-GTP in the presence or absence of 3 μM RocA treatment. (f) Toeprinting assay using 10 μM recombinant eIF4A in the presence or absence of 10 μM RocA treatment. (g) Toeprinting assay (top) and RNase I footprinting assay (bottom) using 10 μM recombinant eIF4A with mRNA containing one AGAGAG motif at the middle in the presence or absence of 10 μM RocA treatment. (h and i) Toeprinting assay using 10 μM recombinant eIF4A (VX 4 GKT) or (D296A-T298K) with mRNA containing seven AGAGAG motifs in the presence or absence of 10 μM RocA treatment. (j) Pre-formation of the complex with RocA and eIF4A (VX 4 GKT) or (D296A-T298K) on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (k) Basal translation level from mRNA containing seven AGAGAG with the supplementation of recombinant eIF4A. (l) In vitro translation in RRL with mRNAs with single polypurine motif (AGAGAG) insertion at the different positions in 5′ UTR (m) Basal translation level from mRNAs bearing PV IRES and PV IRES with three AGAGAG. In b-c and h-j, data represent mean and S.D. (n = 3).

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: Characterization of toeprinting assay (a) Diagram of the reporters used in this study. (b, and c) In vitro translation in RRL with mRNAs containing seven polypurine motif (AGAGAG) insertions (b) and qPCR from the samples (c). (d) Dideoxy terminated sequencing of RNA by reverse transcription verified the toeprinting product length terminated by 48S ribosomes. (e) Ribosome toeprinting assay performed in RRL in the presence of m7-GTP in the presence or absence of 3 μM RocA treatment. (f) Toeprinting assay using 10 μM recombinant eIF4A in the presence or absence of 10 μM RocA treatment. (g) Toeprinting assay (top) and RNase I footprinting assay (bottom) using 10 μM recombinant eIF4A with mRNA containing one AGAGAG motif at the middle in the presence or absence of 10 μM RocA treatment. (h and i) Toeprinting assay using 10 μM recombinant eIF4A (VX 4 GKT) or (D296A-T298K) with mRNA containing seven AGAGAG motifs in the presence or absence of 10 μM RocA treatment. (j) Pre-formation of the complex with RocA and eIF4A (VX 4 GKT) or (D296A-T298K) on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (k) Basal translation level from mRNA containing seven AGAGAG with the supplementation of recombinant eIF4A. (l) In vitro translation in RRL with mRNAs with single polypurine motif (AGAGAG) insertion at the different positions in 5′ UTR (m) Basal translation level from mRNAs bearing PV IRES and PV IRES with three AGAGAG. In b-c and h-j, data represent mean and S.D. (n = 3).

    Article Snippet: Pulldown assay The lysate of E. coli cells expressing eIF4A WT or eIF4A D296A-T298K proteins from 1 ml culture was prepared as described in “Purification of recombinant eIF4A proteins“ and incubated with 10 μl of HisPur Ni-NTA Magnetic Beads (Thermo Scientific) at 4 °C for 30 min.

    Techniques: Toeprinting Assay, In Vitro, Real-time Polymerase Chain Reaction, Sequencing, Recombinant, Footprinting

    eIF4A/RocA complexes on polypurine motifs block scanning of pre-initiation complex, inducing uORF translation (a) Pre-formation of the complex with RocA and eIF4A on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (b) The supplementation of recombinant eIF4A protein to RRL in vitro transaltion reaction with 10 μM Hipp or 3 μM RocA. (c) In vitro translation in RRL with mRNAs with native PV IRES and that with three polypurine motifs ( Extended Data Figure 9a ). (d) Meta-gene analysis of high-sensitivity transcripts to RocA. Reads are normalized to the sum of mitochondrial footprints reads. Histogram of the position of the first polypurine motif (6-mer) after uORF initiation codon (inset). P value is calculated by Fisher’s exact test. Bin width is 12 nt. (e) Western blot of SBP translated from uORF and downstream major ORF in RRL with 0.03 μM RocA treatment. Quantification of bands normalized to long form with DMSO treatment is shown. For gel source data, see Supplementary Fig. 1 . (f) Schematic representation of RocA-mediated translation control. RocA clamps eIF4A onto mRNA by selective affinity enhancement for a polypurine motif in eIF4F-, cap-, and ATP-independent manners, which then blocks scanning of pre-initiation complex, introducing premature translation from uORF and inhibiting downstream ORF translation. In b and c, data represent mean and S.D. (n = 3).

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: eIF4A/RocA complexes on polypurine motifs block scanning of pre-initiation complex, inducing uORF translation (a) Pre-formation of the complex with RocA and eIF4A on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (b) The supplementation of recombinant eIF4A protein to RRL in vitro transaltion reaction with 10 μM Hipp or 3 μM RocA. (c) In vitro translation in RRL with mRNAs with native PV IRES and that with three polypurine motifs ( Extended Data Figure 9a ). (d) Meta-gene analysis of high-sensitivity transcripts to RocA. Reads are normalized to the sum of mitochondrial footprints reads. Histogram of the position of the first polypurine motif (6-mer) after uORF initiation codon (inset). P value is calculated by Fisher’s exact test. Bin width is 12 nt. (e) Western blot of SBP translated from uORF and downstream major ORF in RRL with 0.03 μM RocA treatment. Quantification of bands normalized to long form with DMSO treatment is shown. For gel source data, see Supplementary Fig. 1 . (f) Schematic representation of RocA-mediated translation control. RocA clamps eIF4A onto mRNA by selective affinity enhancement for a polypurine motif in eIF4F-, cap-, and ATP-independent manners, which then blocks scanning of pre-initiation complex, introducing premature translation from uORF and inhibiting downstream ORF translation. In b and c, data represent mean and S.D. (n = 3).

    Article Snippet: Pulldown assay The lysate of E. coli cells expressing eIF4A WT or eIF4A D296A-T298K proteins from 1 ml culture was prepared as described in “Purification of recombinant eIF4A proteins“ and incubated with 10 μl of HisPur Ni-NTA Magnetic Beads (Thermo Scientific) at 4 °C for 30 min.

    Techniques: Blocking Assay, Recombinant, In Vitro, Western Blot

    Characterization of iCLIP data (a) CBB staining of purified SBP-eIF4A protein in iCLIP procedure. (b) Visualization of RNA-crosslinked with SBP-eIF4A and unknown proteins by 32 P labeling of RNA. We avoided the contamination of RNAs cross-linked to the additional, co-purifying, unknown proteins. (c) Distribution of read length in iCLIP libraries. Avoidance of contaminating RNAs restricted us to short RNAs, which likely correspond to the region of RNA physically protected by eIF4A binding, or footprint (d) Nucleotide bias along the reads in iCLIP libraries. The crosslinking bias for U may underestimate the preference for polypurine motifs. (e) Correlations of iCLIP motif enrichment (4-mer) by different RocA concentrations. (f) Correlations of iCLIP motif enrichment (4-mer) by 3 μM RocA and motif prediction of 0.03 μM RocA effect in RIP-Seq. The motifs shown in Figure 3b are highlighted. ρ: Spearman’s rank correlation.

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: Characterization of iCLIP data (a) CBB staining of purified SBP-eIF4A protein in iCLIP procedure. (b) Visualization of RNA-crosslinked with SBP-eIF4A and unknown proteins by 32 P labeling of RNA. We avoided the contamination of RNAs cross-linked to the additional, co-purifying, unknown proteins. (c) Distribution of read length in iCLIP libraries. Avoidance of contaminating RNAs restricted us to short RNAs, which likely correspond to the region of RNA physically protected by eIF4A binding, or footprint (d) Nucleotide bias along the reads in iCLIP libraries. The crosslinking bias for U may underestimate the preference for polypurine motifs. (e) Correlations of iCLIP motif enrichment (4-mer) by different RocA concentrations. (f) Correlations of iCLIP motif enrichment (4-mer) by 3 μM RocA and motif prediction of 0.03 μM RocA effect in RIP-Seq. The motifs shown in Figure 3b are highlighted. ρ: Spearman’s rank correlation.

    Article Snippet: Pulldown assay The lysate of E. coli cells expressing eIF4A WT or eIF4A D296A-T298K proteins from 1 ml culture was prepared as described in “Purification of recombinant eIF4A proteins“ and incubated with 10 μl of HisPur Ni-NTA Magnetic Beads (Thermo Scientific) at 4 °C for 30 min.

    Techniques: Staining, Purification, Labeling, Binding Assay

    RocA represses translation, targeting to eIF4A (a) Polysome profiling experiments with RocA and PP242 treatments. RocA disrupts polysomes dose-dependently. (b) Western blot of phospho-eIF2α and phospho-4EBP shows that effect of RocA is independent of known translation control targeting to eIFs. Phosphorylation of eIF2α and dephosphorylation of 4EBP were induced by Thapsigargin and PP242, respectively. (c and d) Luciferase reporter assay possessing PTGES3 5′ UTR ( Figure 1c ) with exogenous expression of WT or RocA resistant eIF4A mutants (c) and western blot of endogenous and exogenous eIF4A (d). eIF4A is the main molecular target of RocA. Data represent mean and S.D. (n = 3). (e and f) Correlation of sum of the footprint reads to 13 mitochondrial mRNAs among different conditions (e) and correlation of sum of the footprint reads from cytoplasmic ribosomes to each transcript between biological replicates (f). r is Pearson’s correlation. P value is calculated by Student′s t-test. (g and h) Tile plot of codon periodicity along length of mitochondria footprints (g, left) and mitochondria footprint length distribution (g, right) and codon periodicities of 31 nt mitochondrial footprints among different conditions (h). Footprints with 31-nt length showed most homogenous codon periodicity and this periodicity was retained with RocA treatment, showing that mitochondrial ribosome translates even in high doses of RocA.

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: RocA represses translation, targeting to eIF4A (a) Polysome profiling experiments with RocA and PP242 treatments. RocA disrupts polysomes dose-dependently. (b) Western blot of phospho-eIF2α and phospho-4EBP shows that effect of RocA is independent of known translation control targeting to eIFs. Phosphorylation of eIF2α and dephosphorylation of 4EBP were induced by Thapsigargin and PP242, respectively. (c and d) Luciferase reporter assay possessing PTGES3 5′ UTR ( Figure 1c ) with exogenous expression of WT or RocA resistant eIF4A mutants (c) and western blot of endogenous and exogenous eIF4A (d). eIF4A is the main molecular target of RocA. Data represent mean and S.D. (n = 3). (e and f) Correlation of sum of the footprint reads to 13 mitochondrial mRNAs among different conditions (e) and correlation of sum of the footprint reads from cytoplasmic ribosomes to each transcript between biological replicates (f). r is Pearson’s correlation. P value is calculated by Student′s t-test. (g and h) Tile plot of codon periodicity along length of mitochondria footprints (g, left) and mitochondria footprint length distribution (g, right) and codon periodicities of 31 nt mitochondrial footprints among different conditions (h). Footprints with 31-nt length showed most homogenous codon periodicity and this periodicity was retained with RocA treatment, showing that mitochondrial ribosome translates even in high doses of RocA.

    Article Snippet: Pulldown assay The lysate of E. coli cells expressing eIF4A WT or eIF4A D296A-T298K proteins from 1 ml culture was prepared as described in “Purification of recombinant eIF4A proteins“ and incubated with 10 μl of HisPur Ni-NTA Magnetic Beads (Thermo Scientific) at 4 °C for 30 min.

    Techniques: Western Blot, De-Phosphorylation Assay, Luciferase, Reporter Assay, Expressing

    RNA sequence selectivity is imparted upon eIF4A by RocA causing selective translation repression (a) Histogram of the number of transcripts along translation -fold change by ribosome profiling when cells are treated with 0.03, 0.3, or 3 μM RocA, normalized to the number of mitochondrial footprints. Median -fold change is shown. Bin width is 0.1. (b) MA plot of mean footprint reads between 3 μM RocA treatment and non-treatment normalized to library sizes versus translation -fold change by 3 μM RocA treatment, highlighting high-sensitivity and low-sensitivity mRNAs. (c) The 5′ UTRs of indicated genes were fused to Renilla luciferase and these reporter mRNAs were transfected prior to treatment with RocA as indicated. Data represent mean and standard deviation (S.D.) (n = 3). (d) Correlation of translation -fold change to RIP -fold change with RocA treatment. ρ: Spearman’s rank correlation.

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: RNA sequence selectivity is imparted upon eIF4A by RocA causing selective translation repression (a) Histogram of the number of transcripts along translation -fold change by ribosome profiling when cells are treated with 0.03, 0.3, or 3 μM RocA, normalized to the number of mitochondrial footprints. Median -fold change is shown. Bin width is 0.1. (b) MA plot of mean footprint reads between 3 μM RocA treatment and non-treatment normalized to library sizes versus translation -fold change by 3 μM RocA treatment, highlighting high-sensitivity and low-sensitivity mRNAs. (c) The 5′ UTRs of indicated genes were fused to Renilla luciferase and these reporter mRNAs were transfected prior to treatment with RocA as indicated. Data represent mean and standard deviation (S.D.) (n = 3). (d) Correlation of translation -fold change to RIP -fold change with RocA treatment. ρ: Spearman’s rank correlation.

    Article Snippet: Pulldown assay The lysate of E. coli cells expressing eIF4A WT or eIF4A D296A-T298K proteins from 1 ml culture was prepared as described in “Purification of recombinant eIF4A proteins“ and incubated with 10 μl of HisPur Ni-NTA Magnetic Beads (Thermo Scientific) at 4 °C for 30 min.

    Techniques: Sequencing, Luciferase, Transfection, Standard Deviation

    Motif enrichment by Bind-n-Seq (a) Nucleotide composition in each length of reads in input RNAs for Bind-n-Seq. Input RNAs are random in entire read length. (b) Length distribution of reads from Bind-n-Seq. RNAs bound to eIF4A showed longer length distribution, indicating that eIF4A has preference for longer RNAs. (c) Correlations of 4-mer motif enrichment in Bind-n-Seq by 0.03 μM RocA treatment to that by 0.3 μM RocA treatment. (d) Correlations between 5-mer and 6-mer motif enrichment in Bind-n-Seq by 0.03 μM RocA treatment and motif prediction of 0.03 μM RocA effect in RIP-Seq. ρ: Spearman’s rank correlation. (e) Highest-scoring 5-mer and 6-mer motifs in Bind-n-Seq and RIP-Seq. (f) Cumulative fractions along number of 4-mer motifs ( Figure 2b ) in 5′ UTR are plotted to total, RocA high-sensitivity, and RocA low-sensitivity mRNAs. Significance is calculated by Mann-Whitney U test. (g) Correlations of Bind-n-Seq motif enrichment (5-mer) by eIF4A to that by 0.03 μM RocA treatment. The motifs appeared in RNAs used in Extended Data figure 8 are highlighted. (h) Correlation of Bind-n-Seq motif enrichment (5-mer) by eIF4A to motif prediction of Hipp effect in translation change, which is define as Spearman’s correlation of motif number in 5′ UTR to translation -fold change by Hipp. mRNAs with high affinity motif to eIF4A in 5′ UTR are resistant to Hipp treatment. (i) The correlation between enriched motifs of replicates in Bind-n-Seq with ADP + Pi. ρ: Spearman’s rank correlation.

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: Motif enrichment by Bind-n-Seq (a) Nucleotide composition in each length of reads in input RNAs for Bind-n-Seq. Input RNAs are random in entire read length. (b) Length distribution of reads from Bind-n-Seq. RNAs bound to eIF4A showed longer length distribution, indicating that eIF4A has preference for longer RNAs. (c) Correlations of 4-mer motif enrichment in Bind-n-Seq by 0.03 μM RocA treatment to that by 0.3 μM RocA treatment. (d) Correlations between 5-mer and 6-mer motif enrichment in Bind-n-Seq by 0.03 μM RocA treatment and motif prediction of 0.03 μM RocA effect in RIP-Seq. ρ: Spearman’s rank correlation. (e) Highest-scoring 5-mer and 6-mer motifs in Bind-n-Seq and RIP-Seq. (f) Cumulative fractions along number of 4-mer motifs ( Figure 2b ) in 5′ UTR are plotted to total, RocA high-sensitivity, and RocA low-sensitivity mRNAs. Significance is calculated by Mann-Whitney U test. (g) Correlations of Bind-n-Seq motif enrichment (5-mer) by eIF4A to that by 0.03 μM RocA treatment. The motifs appeared in RNAs used in Extended Data figure 8 are highlighted. (h) Correlation of Bind-n-Seq motif enrichment (5-mer) by eIF4A to motif prediction of Hipp effect in translation change, which is define as Spearman’s correlation of motif number in 5′ UTR to translation -fold change by Hipp. mRNAs with high affinity motif to eIF4A in 5′ UTR are resistant to Hipp treatment. (i) The correlation between enriched motifs of replicates in Bind-n-Seq with ADP + Pi. ρ: Spearman’s rank correlation.

    Article Snippet: Pulldown assay The lysate of E. coli cells expressing eIF4A WT or eIF4A D296A-T298K proteins from 1 ml culture was prepared as described in “Purification of recombinant eIF4A proteins“ and incubated with 10 μl of HisPur Ni-NTA Magnetic Beads (Thermo Scientific) at 4 °C for 30 min.

    Techniques: MANN-WHITNEY