competent escherichia coli cells  (Thermo Fisher)


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    Structured Review

    Thermo Fisher competent escherichia coli cells
    Recovery rates of different concentrations (0.1 and 0.001 ng) of bacterial DNA ( <t>Escherichia</t> coli and Bacteroides fragilis ) spiked into DNA of an intestinal biopsy sample (A) and a stool sample (B) of healthy volunteers. The mean recovery of bacterial DNA is 78.76% (range, 70.18 to 90.20%).
    Competent Escherichia Coli Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Quantification of Intestinal Bacterial Populations by Real-Time PCR with a Universal Primer Set and Minor Groove Binder Probes: a Global Approach to the Enteric Flora"

    Article Title: Quantification of Intestinal Bacterial Populations by Real-Time PCR with a Universal Primer Set and Minor Groove Binder Probes: a Global Approach to the Enteric Flora

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.42.6.2566-2572.2004

    Recovery rates of different concentrations (0.1 and 0.001 ng) of bacterial DNA ( Escherichia coli and Bacteroides fragilis ) spiked into DNA of an intestinal biopsy sample (A) and a stool sample (B) of healthy volunteers. The mean recovery of bacterial DNA is 78.76% (range, 70.18 to 90.20%).
    Figure Legend Snippet: Recovery rates of different concentrations (0.1 and 0.001 ng) of bacterial DNA ( Escherichia coli and Bacteroides fragilis ) spiked into DNA of an intestinal biopsy sample (A) and a stool sample (B) of healthy volunteers. The mean recovery of bacterial DNA is 78.76% (range, 70.18 to 90.20%).

    Techniques Used:

    Number of cells detected by real-time PCR in clinical samples (biopsies) of five healthy controls (patients 1 to 5). (A) Total bacteria (VIC-labeled universal probe). (B, C, and D) Escherichia coli ; Bacteroides , Porphyromonas , and Prevotella ; and Enterobacteriaceae , respectively (FAM-labeled specific probes). Normalized mean values of two independent experiments ± standard deviation are shown.
    Figure Legend Snippet: Number of cells detected by real-time PCR in clinical samples (biopsies) of five healthy controls (patients 1 to 5). (A) Total bacteria (VIC-labeled universal probe). (B, C, and D) Escherichia coli ; Bacteroides , Porphyromonas , and Prevotella ; and Enterobacteriaceae , respectively (FAM-labeled specific probes). Normalized mean values of two independent experiments ± standard deviation are shown.

    Techniques Used: Real-time Polymerase Chain Reaction, Labeling, Standard Deviation

    Related Articles

    Clone Assay:

    Article Title: Quantification of Intestinal Bacterial Populations by Real-Time PCR with a Universal Primer Set and Minor Groove Binder Probes: a Global Approach to the Enteric Flora
    Article Snippet: .. DNA was cloned into competent Escherichia coli cells with the pCR2.1 TOPO TA cloning kit (Invitrogen, Karlsruhe, Germany). .. Plasmid DNA from overnight cultures was prepared with the QIAprep 96 Turbo miniprep kit (Qiagen, Hilden, Germany).

    Article Title: Bacterial Communities of Diverse Drosophila Species: Ecological Context of a Host-Microbe Model System
    Article Snippet: .. Clones were transformed chemically into One Shot TOP10 chemically competent E. coli cells or via electroporation into ElectroMAX DH10B E. coli cells (Invitrogen, Carlsbad, CA) and plated onto agar plates with X-gal and either 50 mg/mL Kanamycin or 50 mg/mL Ampicillin. .. Colony PCR (20 colonies) was used to verify a < 10% insertless rate and ∼1.5 kb insert size.

    Article Title: Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis
    Article Snippet: .. The PCR amplicon was then cloned to pCR2.1 vector using TA cloning kit (Invitrogen, San Deigo, CA), transformed into One Shot TOP10 Chemically Competent E. coli cells (Invitrogen, Carlsbad, CA). .. Single colonies were grown in 5 mL LB medium and plasmid DNA was purified with Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, CA).

    Article Title: Predicting RNA hyper-editing with a novel tool when unambiguous alignment is impossible
    Article Snippet: .. Subsequently, 4 μ L of PCR product was cloned into One Shot®;TOP10 Chemically Competent E. coli cells using Zero Blunt®;TOPO®;PCR Cloning Kit from Invitrogen (catalog number: K2800J10), according to manufacturer’s guidelines. .. A total of 50 μ L solution containing the transformed cells were plated on kanamycin+agar plates.

    Amplification:

    Article Title: Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis
    Article Snippet: .. The PCR amplicon was then cloned to pCR2.1 vector using TA cloning kit (Invitrogen, San Deigo, CA), transformed into One Shot TOP10 Chemically Competent E. coli cells (Invitrogen, Carlsbad, CA). .. Single colonies were grown in 5 mL LB medium and plasmid DNA was purified with Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, CA).

    Article Title: CNGA3: A Target of Spinal Nitric Oxide/cGMP Signaling and Modulator of Inflammatory Pain Hypersensitivity
    Article Snippet: .. The PCR products were eluted from the agarose gel using the QIAEX II Agarose Gel Extraction Kit (Qiagen), cloned into the pCR4-TOPO plasmid vector (Invitrogen), and amplified in One Shot TOP10 Chemically Competent E. coli cells (Invitrogen). .. Then the plasmid-DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen), quantified with a NanoDrop spectrophotometer, and sequenced for verification (LGC Genomics).

    Agarose Gel Electrophoresis:

    Article Title: CNGA3: A Target of Spinal Nitric Oxide/cGMP Signaling and Modulator of Inflammatory Pain Hypersensitivity
    Article Snippet: .. The PCR products were eluted from the agarose gel using the QIAEX II Agarose Gel Extraction Kit (Qiagen), cloned into the pCR4-TOPO plasmid vector (Invitrogen), and amplified in One Shot TOP10 Chemically Competent E. coli cells (Invitrogen). .. Then the plasmid-DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen), quantified with a NanoDrop spectrophotometer, and sequenced for verification (LGC Genomics).

    Electroporation:

    Article Title: Bacterial Communities of Diverse Drosophila Species: Ecological Context of a Host-Microbe Model System
    Article Snippet: .. Clones were transformed chemically into One Shot TOP10 chemically competent E. coli cells or via electroporation into ElectroMAX DH10B E. coli cells (Invitrogen, Carlsbad, CA) and plated onto agar plates with X-gal and either 50 mg/mL Kanamycin or 50 mg/mL Ampicillin. .. Colony PCR (20 colonies) was used to verify a < 10% insertless rate and ∼1.5 kb insert size.

    TA Cloning:

    Article Title: Quantification of Intestinal Bacterial Populations by Real-Time PCR with a Universal Primer Set and Minor Groove Binder Probes: a Global Approach to the Enteric Flora
    Article Snippet: .. DNA was cloned into competent Escherichia coli cells with the pCR2.1 TOPO TA cloning kit (Invitrogen, Karlsruhe, Germany). .. Plasmid DNA from overnight cultures was prepared with the QIAprep 96 Turbo miniprep kit (Qiagen, Hilden, Germany).

    Article Title: Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis
    Article Snippet: .. The PCR amplicon was then cloned to pCR2.1 vector using TA cloning kit (Invitrogen, San Deigo, CA), transformed into One Shot TOP10 Chemically Competent E. coli cells (Invitrogen, Carlsbad, CA). .. Single colonies were grown in 5 mL LB medium and plasmid DNA was purified with Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, CA).

    Article Title: Molecular evidence for sediment nitrogen fixation in a temperate New England estuary
    Article Snippet: .. Cleaned fragments were then immediately inserted into a pCR® 2.1-TOPO® vector using One Shot® TOP10 Chemically Competent E. coli cells and a TOPO TA Cloning® Kit (Invitrogen, Leek, The Netherlands). .. Insert-containing white colonies were randomly selected and sequenced at the GENEWIZ DNA sequencing facility ( http://www.genewiz.com/ ) in Cambridge, MA using M13 forward (−20) and M13 reverse primers.

    DNA Sequencing:

    Article Title: The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation
    Article Snippet: .. Eluted plasmids (10 µL) were used to transform One Shot TOP10 chemically competent E. coli cells (Invitrogen, Carlsbad, CA) for subsequent plasmid preparation (QIAprep Spin Miniprep kit, Qiagen, Valencia, CA) and DNA sequencing. ..

    Polymerase Chain Reaction:

    Article Title: Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis
    Article Snippet: .. The PCR amplicon was then cloned to pCR2.1 vector using TA cloning kit (Invitrogen, San Deigo, CA), transformed into One Shot TOP10 Chemically Competent E. coli cells (Invitrogen, Carlsbad, CA). .. Single colonies were grown in 5 mL LB medium and plasmid DNA was purified with Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, CA).

    Article Title: CNGA3: A Target of Spinal Nitric Oxide/cGMP Signaling and Modulator of Inflammatory Pain Hypersensitivity
    Article Snippet: .. The PCR products were eluted from the agarose gel using the QIAEX II Agarose Gel Extraction Kit (Qiagen), cloned into the pCR4-TOPO plasmid vector (Invitrogen), and amplified in One Shot TOP10 Chemically Competent E. coli cells (Invitrogen). .. Then the plasmid-DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen), quantified with a NanoDrop spectrophotometer, and sequenced for verification (LGC Genomics).

    Article Title: Molecular evidence for sediment nitrogen fixation in a temperate New England estuary
    Article Snippet: .. Cleaned fragments were then immediately inserted into a pCR® 2.1-TOPO® vector using One Shot® TOP10 Chemically Competent E. coli cells and a TOPO TA Cloning® Kit (Invitrogen, Leek, The Netherlands). .. Insert-containing white colonies were randomly selected and sequenced at the GENEWIZ DNA sequencing facility ( http://www.genewiz.com/ ) in Cambridge, MA using M13 forward (−20) and M13 reverse primers.

    Article Title: Predicting RNA hyper-editing with a novel tool when unambiguous alignment is impossible
    Article Snippet: .. Subsequently, 4 μ L of PCR product was cloned into One Shot®;TOP10 Chemically Competent E. coli cells using Zero Blunt®;TOPO®;PCR Cloning Kit from Invitrogen (catalog number: K2800J10), according to manufacturer’s guidelines. .. A total of 50 μ L solution containing the transformed cells were plated on kanamycin+agar plates.

    Transformation Assay:

    Article Title: Bacterial Communities of Diverse Drosophila Species: Ecological Context of a Host-Microbe Model System
    Article Snippet: .. Clones were transformed chemically into One Shot TOP10 chemically competent E. coli cells or via electroporation into ElectroMAX DH10B E. coli cells (Invitrogen, Carlsbad, CA) and plated onto agar plates with X-gal and either 50 mg/mL Kanamycin or 50 mg/mL Ampicillin. .. Colony PCR (20 colonies) was used to verify a < 10% insertless rate and ∼1.5 kb insert size.

    Article Title: Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis
    Article Snippet: .. The PCR amplicon was then cloned to pCR2.1 vector using TA cloning kit (Invitrogen, San Deigo, CA), transformed into One Shot TOP10 Chemically Competent E. coli cells (Invitrogen, Carlsbad, CA). .. Single colonies were grown in 5 mL LB medium and plasmid DNA was purified with Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, CA).

    Plasmid Preparation:

    Article Title: Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis
    Article Snippet: .. The PCR amplicon was then cloned to pCR2.1 vector using TA cloning kit (Invitrogen, San Deigo, CA), transformed into One Shot TOP10 Chemically Competent E. coli cells (Invitrogen, Carlsbad, CA). .. Single colonies were grown in 5 mL LB medium and plasmid DNA was purified with Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, CA).

    Article Title: CNGA3: A Target of Spinal Nitric Oxide/cGMP Signaling and Modulator of Inflammatory Pain Hypersensitivity
    Article Snippet: .. The PCR products were eluted from the agarose gel using the QIAEX II Agarose Gel Extraction Kit (Qiagen), cloned into the pCR4-TOPO plasmid vector (Invitrogen), and amplified in One Shot TOP10 Chemically Competent E. coli cells (Invitrogen). .. Then the plasmid-DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen), quantified with a NanoDrop spectrophotometer, and sequenced for verification (LGC Genomics).

    Article Title: Molecular evidence for sediment nitrogen fixation in a temperate New England estuary
    Article Snippet: .. Cleaned fragments were then immediately inserted into a pCR® 2.1-TOPO® vector using One Shot® TOP10 Chemically Competent E. coli cells and a TOPO TA Cloning® Kit (Invitrogen, Leek, The Netherlands). .. Insert-containing white colonies were randomly selected and sequenced at the GENEWIZ DNA sequencing facility ( http://www.genewiz.com/ ) in Cambridge, MA using M13 forward (−20) and M13 reverse primers.

    Article Title: The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation
    Article Snippet: .. Eluted plasmids (10 µL) were used to transform One Shot TOP10 chemically competent E. coli cells (Invitrogen, Carlsbad, CA) for subsequent plasmid preparation (QIAprep Spin Miniprep kit, Qiagen, Valencia, CA) and DNA sequencing. ..

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    Thermo Fisher shotmach1 phage resistant competente coli thermofisher c862003 mycobacterium tuberculosis erdman manzanillo
    Shotmach1 Phage Resistant Competente Coli Thermofisher C862003 Mycobacterium Tuberculosis Erdman Manzanillo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    Image Search Results


    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