rnase t1  (Worthington Biochemical)


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  • 93
    Name:
    Ribonuclease T1 Chromatographically Purif Lyophilized
    Description:
    Highly purified microbial non mammalian RNase prepared with non animal components Supplied as a lyophilized powder
    Catalog Number:
    ls01490
    Price:
    151
    Size:
    500 ku
    Source:
    Aspergillus oryzae
    Cas Number:
    9026.12.4
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    Structured Review

    Worthington Biochemical rnase t1
    Components of the S. pombe <t>RNase</t> MRP complex. (A) RNA component of S. pombe RNase MRP ( mrp1 ). RNA was separated from the purified RNase MRP with acid-phenol treatment and subjected to 8 M urea-7.5% PAGE (SYBR Gold staining). (B) Nucleotide sequence of S. pombe mrp1 RNA and the fragments used for the sequence analysis. Solid or dashed double-headed arrows show the fragments obtained by digestion with <t>RNase</t> T1 or MazF/PemK RNase, respectively. RNase T1 fragments were identified by Ariadne search program, and PemK/MazF fragments were identified by manual inspection of MS/MS spectra (see also Table S2 ). m 3 Gppp, trimethylguanosine cap. (C) Protein components of S. pombe RNase MRP. The RNase-MRP preparation affinity-purified using FEM3-tagged Rmp1 as bait (Rmp1-FEM3) was separated by SDS-PAGE and visualized with Coomassie Brilliant Blue staining. The proteins identified by LC-MS/MS are shown on the right (see also Table S3 and Figure S6 ). The IgG probably resulted from sloughing from the beads during the affinity purification.
    Highly purified microbial non mammalian RNase prepared with non animal components Supplied as a lyophilized powder
    https://www.bioz.com/result/rnase t1/product/Worthington Biochemical
    Average 93 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    rnase t1 - by Bioz Stars, 2021-02
    93/100 stars

    Images

    1) Product Images from "RNase MRP Cleaves Pre-tRNASer-Met in the tRNA Maturation Pathway"

    Article Title: RNase MRP Cleaves Pre-tRNASer-Met in the tRNA Maturation Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112488

    Components of the S. pombe RNase MRP complex. (A) RNA component of S. pombe RNase MRP ( mrp1 ). RNA was separated from the purified RNase MRP with acid-phenol treatment and subjected to 8 M urea-7.5% PAGE (SYBR Gold staining). (B) Nucleotide sequence of S. pombe mrp1 RNA and the fragments used for the sequence analysis. Solid or dashed double-headed arrows show the fragments obtained by digestion with RNase T1 or MazF/PemK RNase, respectively. RNase T1 fragments were identified by Ariadne search program, and PemK/MazF fragments were identified by manual inspection of MS/MS spectra (see also Table S2 ). m 3 Gppp, trimethylguanosine cap. (C) Protein components of S. pombe RNase MRP. The RNase-MRP preparation affinity-purified using FEM3-tagged Rmp1 as bait (Rmp1-FEM3) was separated by SDS-PAGE and visualized with Coomassie Brilliant Blue staining. The proteins identified by LC-MS/MS are shown on the right (see also Table S3 and Figure S6 ). The IgG probably resulted from sloughing from the beads during the affinity purification.
    Figure Legend Snippet: Components of the S. pombe RNase MRP complex. (A) RNA component of S. pombe RNase MRP ( mrp1 ). RNA was separated from the purified RNase MRP with acid-phenol treatment and subjected to 8 M urea-7.5% PAGE (SYBR Gold staining). (B) Nucleotide sequence of S. pombe mrp1 RNA and the fragments used for the sequence analysis. Solid or dashed double-headed arrows show the fragments obtained by digestion with RNase T1 or MazF/PemK RNase, respectively. RNase T1 fragments were identified by Ariadne search program, and PemK/MazF fragments were identified by manual inspection of MS/MS spectra (see also Table S2 ). m 3 Gppp, trimethylguanosine cap. (C) Protein components of S. pombe RNase MRP. The RNase-MRP preparation affinity-purified using FEM3-tagged Rmp1 as bait (Rmp1-FEM3) was separated by SDS-PAGE and visualized with Coomassie Brilliant Blue staining. The proteins identified by LC-MS/MS are shown on the right (see also Table S3 and Figure S6 ). The IgG probably resulted from sloughing from the beads during the affinity purification.

    Techniques Used: Purification, Polyacrylamide Gel Electrophoresis, Staining, Sequencing, Mass Spectrometry, Affinity Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy

    Isolation of the catalytically active core of RNase MRP. (A) Urea-PAGE profiles of mrp1 RNA fragments produced by RNase A–mediated limited nucleolysis of RNase MRP (SYBR Gold staining). The S. pombe RNase MRP obtained from a 2-l culture of logarithmically growing cells was digested on FLAG M2 agarose beads at 4°C for 1 h with increasing amounts of RNase A. Lane 1, no RNase A; lane 2, 1 µg/ml; lane 3, 5 µg/ml; lane 4, 10 µg/ml. A portion (5%) of each reaction was loaded per lane. (B) A core of RNase MRP produced by RNase A–mediated limited nucleolysis cleaves pre-tRNA Ser-Met . RNase MRP (pulled down with tagged Rmp1 from JJ095 cells using FLAG M2 agarose) and the mock preparation (pulled down from SP6 cells) were incubated with (+) or without (−) RNase A. Each digested preparation (1 pmol each) was incubated with pre-tRNA Ser-Met (8 pmol) at 37°C for 30 min, and each reaction mixture was subjected to urea-PAGE (SYBR-Gold staining). Band 1, mrp1 RNA; 2, pre-tRNA Ser-Met ; 3, pre-tRNA Ser +trailer; 4, tRNA Met . (C) Double-reciprocal plot of the catalytic reaction of RNase A–mediated partial nucleolysis of RNase MRP. The reaction was performed for 30 min with synthetic pre-tRNA Ser-Met as a substrate under the conditions as in Figure 3B . The plot indicates K M of 0.974 µM and Vmax of 12.9 nM/min. (D) RNA fragments produced by RNase T1 digestion of Band 1 (indicated by solid lines) and Band 2 (broken lines) in (A). The fragments identified by LC-MS/MS are mapped on the mrp1 RNA sequence, where the conserved helices and strands of mrp1 [1] , [29] , [31] are shown in shaded boxes. (E) Nuclease-resistant regions mapped in the secondary structure of mrp1 . The map was according to the previous study [2] with modifications made by the assistance of CentroidHomFold ( http://www.ncrna.org/centroidhomfold ). Dashed-line boxes denote the two putative domains [2] . The nuclease-resistant region is shaded gray. The nucleotides with dotted bar are the consensus sequence ANAGNNA known as the mCR-IV motif [2] . (F) SDS-PAGE profiles of the protein components of RNase MRP before (−) and after (+) RNase A–mediated limited nucleolysis (Coomassie Brilliant Blue staining). The proteins assigned by LC-MS/MS are also shown. Note that the catalytic core of RNase MRP produced by partial nucleolysis contains 8 (underlined) of the 11 total subunits.
    Figure Legend Snippet: Isolation of the catalytically active core of RNase MRP. (A) Urea-PAGE profiles of mrp1 RNA fragments produced by RNase A–mediated limited nucleolysis of RNase MRP (SYBR Gold staining). The S. pombe RNase MRP obtained from a 2-l culture of logarithmically growing cells was digested on FLAG M2 agarose beads at 4°C for 1 h with increasing amounts of RNase A. Lane 1, no RNase A; lane 2, 1 µg/ml; lane 3, 5 µg/ml; lane 4, 10 µg/ml. A portion (5%) of each reaction was loaded per lane. (B) A core of RNase MRP produced by RNase A–mediated limited nucleolysis cleaves pre-tRNA Ser-Met . RNase MRP (pulled down with tagged Rmp1 from JJ095 cells using FLAG M2 agarose) and the mock preparation (pulled down from SP6 cells) were incubated with (+) or without (−) RNase A. Each digested preparation (1 pmol each) was incubated with pre-tRNA Ser-Met (8 pmol) at 37°C for 30 min, and each reaction mixture was subjected to urea-PAGE (SYBR-Gold staining). Band 1, mrp1 RNA; 2, pre-tRNA Ser-Met ; 3, pre-tRNA Ser +trailer; 4, tRNA Met . (C) Double-reciprocal plot of the catalytic reaction of RNase A–mediated partial nucleolysis of RNase MRP. The reaction was performed for 30 min with synthetic pre-tRNA Ser-Met as a substrate under the conditions as in Figure 3B . The plot indicates K M of 0.974 µM and Vmax of 12.9 nM/min. (D) RNA fragments produced by RNase T1 digestion of Band 1 (indicated by solid lines) and Band 2 (broken lines) in (A). The fragments identified by LC-MS/MS are mapped on the mrp1 RNA sequence, where the conserved helices and strands of mrp1 [1] , [29] , [31] are shown in shaded boxes. (E) Nuclease-resistant regions mapped in the secondary structure of mrp1 . The map was according to the previous study [2] with modifications made by the assistance of CentroidHomFold ( http://www.ncrna.org/centroidhomfold ). Dashed-line boxes denote the two putative domains [2] . The nuclease-resistant region is shaded gray. The nucleotides with dotted bar are the consensus sequence ANAGNNA known as the mCR-IV motif [2] . (F) SDS-PAGE profiles of the protein components of RNase MRP before (−) and after (+) RNase A–mediated limited nucleolysis (Coomassie Brilliant Blue staining). The proteins assigned by LC-MS/MS are also shown. Note that the catalytic core of RNase MRP produced by partial nucleolysis contains 8 (underlined) of the 11 total subunits.

    Techniques Used: Isolation, Polyacrylamide Gel Electrophoresis, Produced, Staining, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, SDS Page

    2) Product Images from "Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile"

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1009

    Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.
    Figure Legend Snippet: Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.

    Techniques Used: Binding Assay, Purification, Mutagenesis, Isolation, Concentration Assay

    Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.
    Figure Legend Snippet: Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.

    Techniques Used: Purification, Mutagenesis, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Clear Native PAGE, Staining, Northern Blot, Hybridization

    3) Product Images from "Structural and mechanistic basis for enhanced translational efficiency by 2-thiouridine at the tRNA anticodon wobble position"

    Article Title: Structural and mechanistic basis for enhanced translational efficiency by 2-thiouridine at the tRNA anticodon wobble position

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2013.05.018

    A: The total ion chromatogram (TIC) of RNase T1 digested E. coli tRNA Gln and the accompanying extracted ion chromatogram (XIC) of m/z 1004, which corresponds to the expected m/z for the fragment with the sequence U[Um]U[cmnm 5 s 2 U]UGp. B: The MS spectrum of the eluent at 28.2 min. depicting the m/z 1004.25, consistent with the sequence U[Um]U[cmnm 5 s 2 U]UGp. C: The peak in B was selected for MS/MS analysis. The resulting fragmentation is consistent with U[Um]U[cmnm 5 s 2 U]UGp with all identified c- and y-type product ions labeled.
    Figure Legend Snippet: A: The total ion chromatogram (TIC) of RNase T1 digested E. coli tRNA Gln and the accompanying extracted ion chromatogram (XIC) of m/z 1004, which corresponds to the expected m/z for the fragment with the sequence U[Um]U[cmnm 5 s 2 U]UGp. B: The MS spectrum of the eluent at 28.2 min. depicting the m/z 1004.25, consistent with the sequence U[Um]U[cmnm 5 s 2 U]UGp. C: The peak in B was selected for MS/MS analysis. The resulting fragmentation is consistent with U[Um]U[cmnm 5 s 2 U]UGp with all identified c- and y-type product ions labeled.

    Techniques Used: Sequencing, Mass Spectrometry, Labeling

    4) Product Images from "Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile"

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1009

    Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.
    Figure Legend Snippet: Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.

    Techniques Used: Binding Assay, Purification, Mutagenesis, Isolation, Concentration Assay

    Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.
    Figure Legend Snippet: Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.

    Techniques Used: Purification, Mutagenesis, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Clear Native PAGE, Staining, Northern Blot, Hybridization

    5) Product Images from "Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA"

    Article Title: Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp782

    RBM15 and OTT3 are required for general mRNA export. Human 293 cells were transfected with siRNAs targeting RBM15, OTT3 or non-targeting siRNA ( control ) and analyzed at day 2 or 4 posttransfection as indicated. ( A ) RT–qPCR detection of RBM15 and OTT3 transcripts at day 2. Expression levels were calculated from real-time PCR values ( C t ) using relative quantitation method and are plotted on the y -axis after normalization to those obtained in the cells transfected with the non-targeting siRNA control (normalized expression). Mean ( n =3) values are presented, and bars show one SEM. ( B ) Cells were transfected with the indicated siRNAs and, next day, with a plasmid expressing HA-OTT3 (1 μg). At day 2 or day 4 after siRNA transfection, cell pellets were boiled in Laemmli sample buffer, proteins separated on 10% SDS–PAGE and analyzed on western blots with antibodies to RBM15, HA, Ran, β-actin or SR proteins as indicated; or by Coomassie staining. ( C ) Cells at day 2 (left panel) or day 4 (right panel) posttransfection were separated into the C, N1 and N2 fractions, mRNA poly(A) tails were 3′-radiolabeled, cut off by RNase T1 digestion, separated by urea–PAGE and detected by phosphoimager. Positions of size markers (nt) are shown. ( D ) U snRNAs from the same fractions as in (D) were separated by urea–PAGE and detected on northern blots, and positions of the individual U snRNAs are indicated to the left. tRNA was detected on the same gels, by ethidium bromide staining prior to blotting (tRNA).
    Figure Legend Snippet: RBM15 and OTT3 are required for general mRNA export. Human 293 cells were transfected with siRNAs targeting RBM15, OTT3 or non-targeting siRNA ( control ) and analyzed at day 2 or 4 posttransfection as indicated. ( A ) RT–qPCR detection of RBM15 and OTT3 transcripts at day 2. Expression levels were calculated from real-time PCR values ( C t ) using relative quantitation method and are plotted on the y -axis after normalization to those obtained in the cells transfected with the non-targeting siRNA control (normalized expression). Mean ( n =3) values are presented, and bars show one SEM. ( B ) Cells were transfected with the indicated siRNAs and, next day, with a plasmid expressing HA-OTT3 (1 μg). At day 2 or day 4 after siRNA transfection, cell pellets were boiled in Laemmli sample buffer, proteins separated on 10% SDS–PAGE and analyzed on western blots with antibodies to RBM15, HA, Ran, β-actin or SR proteins as indicated; or by Coomassie staining. ( C ) Cells at day 2 (left panel) or day 4 (right panel) posttransfection were separated into the C, N1 and N2 fractions, mRNA poly(A) tails were 3′-radiolabeled, cut off by RNase T1 digestion, separated by urea–PAGE and detected by phosphoimager. Positions of size markers (nt) are shown. ( D ) U snRNAs from the same fractions as in (D) were separated by urea–PAGE and detected on northern blots, and positions of the individual U snRNAs are indicated to the left. tRNA was detected on the same gels, by ethidium bromide staining prior to blotting (tRNA).

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, Plasmid Preparation, SDS Page, Western Blot, Staining, Polyacrylamide Gel Electrophoresis, Northern Blot

    6) Product Images from "Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile"

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1009

    Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.
    Figure Legend Snippet: Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.

    Techniques Used: Binding Assay, Purification, Mutagenesis, Isolation, Concentration Assay

    Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.
    Figure Legend Snippet: Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.

    Techniques Used: Purification, Mutagenesis, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Clear Native PAGE, Staining, Northern Blot, Hybridization

    7) Product Images from "Identification of the mass-silent post-transcriptionally modified nucleoside pseudouridine in RNA by matrix-assisted laser desorption/ionization mass spectrometry"

    Article Title: Identification of the mass-silent post-transcriptionally modified nucleoside pseudouridine in RNA by matrix-assisted laser desorption/ionization mass spectrometry

    Journal: Nucleic Acids Research

    doi:

    Reverse phase HPLC chromatograms of ( A ) control RNase T1 digest of tRNA Phe and ( B ) RNase T1 digest of CMC-reacted tRNA Phe . The three chromatographic peaks at 13.4, 16.4 and 30.0 min in the bottom chromatogram have absorbances that are 2–5 times greater than their counterparts in the control chromatogram.
    Figure Legend Snippet: Reverse phase HPLC chromatograms of ( A ) control RNase T1 digest of tRNA Phe and ( B ) RNase T1 digest of CMC-reacted tRNA Phe . The three chromatographic peaks at 13.4, 16.4 and 30.0 min in the bottom chromatogram have absorbances that are 2–5 times greater than their counterparts in the control chromatogram.

    Techniques Used: High Performance Liquid Chromatography

    8) Product Images from "A mass spectrometry-based method for direct determination of pseudouridine in RNA"

    Article Title: A mass spectrometry-based method for direct determination of pseudouridine in RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1462

    Identification of four Ψ-containing fragments in human U6 snRNA. The RNase T1 digest of human U6 snRNA (100 fmol) was analyzed by LC-MS and MS 2 . The base peak chromatogram (BPC) of MS, EPIC of MS 2 and four EICs of MS are shown. The major peaks in the BPC, indicated by the arrow or arrowheads with peak numbers, were assigned to the fragments of U6 snRNA (see Supplementary Table S1). The peaks in the EPIC indicated by asterisks were assigned to the Ψ-containing fragments of U6 snRNA by Ariadne, whereas the peaks with numbered closed circles indicate fragments from contaminating RNAs in our U6 snRNA preparation and the U6 snRNA fragment with the 2′,3′-cyclic-phosphate terminus assigned as follows: 1, CΨUCG > p (U6 snRNA); 2, TΨCGp (tRNA); 3, AUΨUCCGp (U5 snRNA); 4, A(Cm)UAAAGp (U5 snRNA); 5, CAUAAAUCUUUC(Gm)CCUUU(Um)A(Cm)ΨAAAGp (U5 snRNA); 6, UAUAAAUCUUUC(Gm)CCUUU(Um)A(Cm)ΨAAAGp (U5 snRNA). The oligoribonucleotide with a sequence UUCCGp detected in the EIC with m/z 791.58 was derived from U5 snRNA contaminated in our U6 snRNA preparation. Note that contaminated RNA fragments can easily be distinguished those derived from a sample RNA by comparing the identified sequence with its genomic sequence using Ariadne. The peaks of the Ψ-containing fragments of U6 snRNA reappear in the EIC. The sequence of each RNase T1 fragment and its m/z value (±15 ppm mass tolerance) for extraction are indicated on each EIC. Corresponding peaks on the EPIC and EIC are linked with dotted lines.
    Figure Legend Snippet: Identification of four Ψ-containing fragments in human U6 snRNA. The RNase T1 digest of human U6 snRNA (100 fmol) was analyzed by LC-MS and MS 2 . The base peak chromatogram (BPC) of MS, EPIC of MS 2 and four EICs of MS are shown. The major peaks in the BPC, indicated by the arrow or arrowheads with peak numbers, were assigned to the fragments of U6 snRNA (see Supplementary Table S1). The peaks in the EPIC indicated by asterisks were assigned to the Ψ-containing fragments of U6 snRNA by Ariadne, whereas the peaks with numbered closed circles indicate fragments from contaminating RNAs in our U6 snRNA preparation and the U6 snRNA fragment with the 2′,3′-cyclic-phosphate terminus assigned as follows: 1, CΨUCG > p (U6 snRNA); 2, TΨCGp (tRNA); 3, AUΨUCCGp (U5 snRNA); 4, A(Cm)UAAAGp (U5 snRNA); 5, CAUAAAUCUUUC(Gm)CCUUU(Um)A(Cm)ΨAAAGp (U5 snRNA); 6, UAUAAAUCUUUC(Gm)CCUUU(Um)A(Cm)ΨAAAGp (U5 snRNA). The oligoribonucleotide with a sequence UUCCGp detected in the EIC with m/z 791.58 was derived from U5 snRNA contaminated in our U6 snRNA preparation. Note that contaminated RNA fragments can easily be distinguished those derived from a sample RNA by comparing the identified sequence with its genomic sequence using Ariadne. The peaks of the Ψ-containing fragments of U6 snRNA reappear in the EIC. The sequence of each RNase T1 fragment and its m/z value (±15 ppm mass tolerance) for extraction are indicated on each EIC. Corresponding peaks on the EPIC and EIC are linked with dotted lines.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Derivative Assay

    Identification of four Ψ-containing fragments in human U5 snRNA. The RNase T1 digest of human U5 snRNA (100 fmol mixture of U5A and U5B) was analyzed by LC-MS and MS 2 . The BPC of MS, EPIC of MS 2 and four EICs of MS are shown. The major peaks in the BPC, indicated by the arrow or arrowheads with peak numbers, were assigned to the fragments of U5A or U5B snRNA (see Supplementary Table S1). The major peaks in EPIC indicated by asterisks were assigned to the Ψ-containing fragments of U5A/U5B snRNA. The peaks with numbered closed circles in the EPIC were fragments of contaminating RNA in our U5A/B preparation assigned as follows: 1, CΨUCGp (U6 snRNA); 2, TΨCGp (tRNA); 3, ΨAC(mA)Gp (U6 snRNA). The peaks of Ψ-containing fragments of U5A or U5B snRNA reappear in the EIC. The sequence of each RNase T1 fragment and its m/z value (±15 ppm mass tolerance) are indicated. Corresponding peaks on EPIC and EIC are linked with dotted lines.
    Figure Legend Snippet: Identification of four Ψ-containing fragments in human U5 snRNA. The RNase T1 digest of human U5 snRNA (100 fmol mixture of U5A and U5B) was analyzed by LC-MS and MS 2 . The BPC of MS, EPIC of MS 2 and four EICs of MS are shown. The major peaks in the BPC, indicated by the arrow or arrowheads with peak numbers, were assigned to the fragments of U5A or U5B snRNA (see Supplementary Table S1). The major peaks in EPIC indicated by asterisks were assigned to the Ψ-containing fragments of U5A/U5B snRNA. The peaks with numbered closed circles in the EPIC were fragments of contaminating RNA in our U5A/B preparation assigned as follows: 1, CΨUCGp (U6 snRNA); 2, TΨCGp (tRNA); 3, ΨAC(mA)Gp (U6 snRNA). The peaks of Ψ-containing fragments of U5A or U5B snRNA reappear in the EIC. The sequence of each RNase T1 fragment and its m/z value (±15 ppm mass tolerance) are indicated. Corresponding peaks on EPIC and EIC are linked with dotted lines.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing

    9) Product Images from "Improved RNA modification mapping of cellular non-coding RNAs using C- and U-specific RNases"

    Article Title: Improved RNA modification mapping of cellular non-coding RNAs using C- and U-specific RNases

    Journal: The Analyst

    doi: 10.1039/c9an02111f

    A portion of 23S rRNA sequence matched against the complementary digestion products of RNases T1, cusativin and MC1. Note the sequence overlaps observed between the digestion products of RNase T1 (red colored font), cusativin (green), and MC1 (blue). The digestion products depicted in brown did not generate the MS/MS spectrum. However, their m/z values exhibited mass error of less than 5 ppm.
    Figure Legend Snippet: A portion of 23S rRNA sequence matched against the complementary digestion products of RNases T1, cusativin and MC1. Note the sequence overlaps observed between the digestion products of RNase T1 (red colored font), cusativin (green), and MC1 (blue). The digestion products depicted in brown did not generate the MS/MS spectrum. However, their m/z values exhibited mass error of less than 5 ppm.

    Techniques Used: Sequencing, Tandem Mass Spectroscopy

    10) Product Images from "The complete chemical structure of Saccharomyces cerevisiae rRNA: partial pseudouridylation of U2345 in 25S rRNA by snoRNA snR9"

    Article Title: The complete chemical structure of Saccharomyces cerevisiae rRNA: partial pseudouridylation of U2345 in 25S rRNA by snoRNA snR9

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw564

    Nucleotide heterogeneity at position 2345 in Saccharomyces cerevisiae 25S rRNA. LC-MS and MS 2 of the RNase T1 digest of U/C-5D-labelled and reverse-phase LC-purified 25S rRNA (100 fmol). ( A ) Extracted ion monitoring of fragments containing a nt at position 2345 shows that nt2345 is a mixture containing 9% U, 74% Um, 4% Ψ and 13% Ψm (Table 2 ). The extracted ion masses have m/z values of 1421.688 (upper panel) and 1418.184 (lower panel) within a mass window of ±10 ppm. U, 2334 UCmCCΨAUCUAC U AΨCΨA 2351 Gp; Ψ, 2334 UCmCCΨAUCUAC Ψ AΨCΨA 2351 Gp; Um, 2334 UCmCCΨAUCUAC Um AΨCΨA 2351 Gp; Ψm, 2334 UCmCCΨAUCUAC Ψm AΨCΨA 2351 Gp. ( B ) Mass spectra for the oligonucleotides containing U2345. The most abundant isotopomers are marked by asterisks. The m/z values of the most abundant signals coincide with the theoretical values within 5 ppm. ( C ) Tandem mass spectrum of 2334 UCmCCΨAUCUACΨmAΨCΨA 2351 Gp. The blue arrows in the spectrum identify the major a, c, w and y ions. The cleavage positions of the assigned ions are mapped on the RNA sequence in the inset. Errors determined by Ariadne in the MS 2 signals are plotted under the spectrum.
    Figure Legend Snippet: Nucleotide heterogeneity at position 2345 in Saccharomyces cerevisiae 25S rRNA. LC-MS and MS 2 of the RNase T1 digest of U/C-5D-labelled and reverse-phase LC-purified 25S rRNA (100 fmol). ( A ) Extracted ion monitoring of fragments containing a nt at position 2345 shows that nt2345 is a mixture containing 9% U, 74% Um, 4% Ψ and 13% Ψm (Table 2 ). The extracted ion masses have m/z values of 1421.688 (upper panel) and 1418.184 (lower panel) within a mass window of ±10 ppm. U, 2334 UCmCCΨAUCUAC U AΨCΨA 2351 Gp; Ψ, 2334 UCmCCΨAUCUAC Ψ AΨCΨA 2351 Gp; Um, 2334 UCmCCΨAUCUAC Um AΨCΨA 2351 Gp; Ψm, 2334 UCmCCΨAUCUAC Ψm AΨCΨA 2351 Gp. ( B ) Mass spectra for the oligonucleotides containing U2345. The most abundant isotopomers are marked by asterisks. The m/z values of the most abundant signals coincide with the theoretical values within 5 ppm. ( C ) Tandem mass spectrum of 2334 UCmCCΨAUCUACΨmAΨCΨA 2351 Gp. The blue arrows in the spectrum identify the major a, c, w and y ions. The cleavage positions of the assigned ions are mapped on the RNA sequence in the inset. Errors determined by Ariadne in the MS 2 signals are plotted under the spectrum.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Purification, Sequencing

    11) Product Images from "Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile"

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1009

    Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.
    Figure Legend Snippet: Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.

    Techniques Used: Binding Assay, Purification, Mutagenesis, Isolation, Concentration Assay

    Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.
    Figure Legend Snippet: Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.

    Techniques Used: Purification, Mutagenesis, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Clear Native PAGE, Staining, Northern Blot, Hybridization

    12) Product Images from "Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF *"

    Article Title: Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.747865

    CID-based sequencing of the double carbodiimide-tagged RNase T1 digestion product from the anticodon of E. coli tRNA Tyr(QUA) . Shown is the CID MS/MS spectrum of ACU[Q]UA[ms 2 i 6 A]AΨCUG with two carbodiimide units (+502 Da, m / z 1506.6). The appearance
    Figure Legend Snippet: CID-based sequencing of the double carbodiimide-tagged RNase T1 digestion product from the anticodon of E. coli tRNA Tyr(QUA) . Shown is the CID MS/MS spectrum of ACU[Q]UA[ms 2 i 6 A]AΨCUG with two carbodiimide units (+502 Da, m / z 1506.6). The appearance

    Techniques Used: Sequencing, Mass Spectrometry

    LC-MS analysis of the carbodiimide-tagged RNase T1 digestion product the from anticodon of E. coli tRNA Tyr(QUA) . A , total ion chromatogram representing the elution pattern of all the oligonucleotide digestion products detected as anions. B , XIC for m
    Figure Legend Snippet: LC-MS analysis of the carbodiimide-tagged RNase T1 digestion product the from anticodon of E. coli tRNA Tyr(QUA) . A , total ion chromatogram representing the elution pattern of all the oligonucleotide digestion products detected as anions. B , XIC for m

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    CID-based sequencing of the single carbodiimide-tagged RNase T1 digestion product the from anticodon of E. coli tRNA Tyr(QUA) . Shown is the CID MS/MS spectrum of ACU[Q]UA[ms 2 i 6 A]AΨCUG with one carbodiimide ( m / z 1422.8) tagged at either one of two
    Figure Legend Snippet: CID-based sequencing of the single carbodiimide-tagged RNase T1 digestion product the from anticodon of E. coli tRNA Tyr(QUA) . Shown is the CID MS/MS spectrum of ACU[Q]UA[ms 2 i 6 A]AΨCUG with one carbodiimide ( m / z 1422.8) tagged at either one of two

    Techniques Used: Sequencing, Mass Spectrometry

    LC-MS analysis of acrylonitrile-derivatized RNase T1/BAP digests of E. coli 23S rRNA. A , XIC for m / z 1305.2 corresponding to [Ψ][Ψ]CG with two cyanoethylations. B , mass spectrum corresponding to XIC peak at 23.4 min showing the presence
    Figure Legend Snippet: LC-MS analysis of acrylonitrile-derivatized RNase T1/BAP digests of E. coli 23S rRNA. A , XIC for m / z 1305.2 corresponding to [Ψ][Ψ]CG with two cyanoethylations. B , mass spectrum corresponding to XIC peak at 23.4 min showing the presence

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    LC-MS/MS Analysis of RNase T1 Digest of E. coli tRNATyr
    Figure Legend Snippet: LC-MS/MS Analysis of RNase T1 Digest of E. coli tRNATyr

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    CID-based sequencing of the acrylonitrile-tagged RNase T1 digestion product the from anticodon of E. coli tDNA Tyr transcript. A , MS/MS spectrum of the doubly charged oligonucleotide ion originating from tDNA Tyr transcript treated with recombinant RluF
    Figure Legend Snippet: CID-based sequencing of the acrylonitrile-tagged RNase T1 digestion product the from anticodon of E. coli tDNA Tyr transcript. A , MS/MS spectrum of the doubly charged oligonucleotide ion originating from tDNA Tyr transcript treated with recombinant RluF

    Techniques Used: Sequencing, Mass Spectrometry, Recombinant

    13) Product Images from "RNA Cytidine Acetyltransferase of Small-Subunit Ribosomal RNA: Identification of Acetylation Sites and the Responsible Acetyltransferase in Fission Yeast, Schizosaccharomyces pombe"

    Article Title: RNA Cytidine Acetyltransferase of Small-Subunit Ribosomal RNA: Identification of Acetylation Sites and the Responsible Acetyltransferase in Fission Yeast, Schizosaccharomyces pombe

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112156

    Effect of Nat10 on the acetylation of cytidines 1297 and 1815 in 18S rRNA. Extracted ion monitoring of RNase T1 fragments of 18S rRNA containing AcC-1297 (upper panel) and AcC-1815 (lower panel) is shown. The analysis was performed for 18S rRNAs purified from strain SP6 supplemented with the mock vector, strain Nat10_G285D supplemented with the mock vector, or strain Nat10_G285D supplemented with pREP1-Nat10_wt, respectively, as indicated. Each yeast strain was grown at 30°C in EMMmedium without leucine. The rRNA was digested by RNase T1 and applied to the LC-MS system (50 fmol each). The sequences and m/z values of AcC-containing nucleotides are indicated. A mass window of 3 ppm was used for the extractions. Y axis indicates the peak intensity relative to the most intensive peak in each panel. Note that the MS signals of AcC-containing nucleotides at the positions indicated by arrows appear in the Nat10_G285D mutant strain upon expression of Nat10 cDNA.
    Figure Legend Snippet: Effect of Nat10 on the acetylation of cytidines 1297 and 1815 in 18S rRNA. Extracted ion monitoring of RNase T1 fragments of 18S rRNA containing AcC-1297 (upper panel) and AcC-1815 (lower panel) is shown. The analysis was performed for 18S rRNAs purified from strain SP6 supplemented with the mock vector, strain Nat10_G285D supplemented with the mock vector, or strain Nat10_G285D supplemented with pREP1-Nat10_wt, respectively, as indicated. Each yeast strain was grown at 30°C in EMMmedium without leucine. The rRNA was digested by RNase T1 and applied to the LC-MS system (50 fmol each). The sequences and m/z values of AcC-containing nucleotides are indicated. A mass window of 3 ppm was used for the extractions. Y axis indicates the peak intensity relative to the most intensive peak in each panel. Note that the MS signals of AcC-containing nucleotides at the positions indicated by arrows appear in the Nat10_G285D mutant strain upon expression of Nat10 cDNA.

    Techniques Used: Purification, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Mutagenesis, Expressing

    MS-based identification of acetylcytidines in S. pombe 18S rRNA. A. Extracted ion monitoring of RNase T1 fragments of 18S rRNA containing acetylcytidine (AcC) and cytidine (C)-1297 (upper panel) and AcC and C-1815 (lower panel). The 18S rRNAs were purified from strain SP6 or Nat10_G285D grown at 30°C in YE medium, digested by RNase T1 and applied to the LC-MS system (50 fmol each). The sequences and m/z values of AcC and C-containing nucleotides are indicated. A mass window of 3 ppm was used for the extractions. Y axis indicates the peak intensity relative to the most intensive peak in each panel. Note that the MS signals of AcC-containing nucleotides were completely lost in the Nat10_G285D mutant strain (indicated by arrows). B. MS/MS spectra of AcC-containing fragments. The acetylated RNA ions [C(AcC)Gp 1− , m/z 1014.14; UUUC(AcC)Gp 2− , m/z 965.60, in A) were analyzed by collision-induced dissociation. The position of acetylcytidine residues was identified by manual interpretation of the a-, c-, w- and y-type series ions and other specific product ions as indicated in the figure. The series ions assigned are indicated on the RNA sequence in the inset.
    Figure Legend Snippet: MS-based identification of acetylcytidines in S. pombe 18S rRNA. A. Extracted ion monitoring of RNase T1 fragments of 18S rRNA containing acetylcytidine (AcC) and cytidine (C)-1297 (upper panel) and AcC and C-1815 (lower panel). The 18S rRNAs were purified from strain SP6 or Nat10_G285D grown at 30°C in YE medium, digested by RNase T1 and applied to the LC-MS system (50 fmol each). The sequences and m/z values of AcC and C-containing nucleotides are indicated. A mass window of 3 ppm was used for the extractions. Y axis indicates the peak intensity relative to the most intensive peak in each panel. Note that the MS signals of AcC-containing nucleotides were completely lost in the Nat10_G285D mutant strain (indicated by arrows). B. MS/MS spectra of AcC-containing fragments. The acetylated RNA ions [C(AcC)Gp 1− , m/z 1014.14; UUUC(AcC)Gp 2− , m/z 965.60, in A) were analyzed by collision-induced dissociation. The position of acetylcytidine residues was identified by manual interpretation of the a-, c-, w- and y-type series ions and other specific product ions as indicated in the figure. The series ions assigned are indicated on the RNA sequence in the inset.

    Techniques Used: Mass Spectrometry, Purification, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Sequencing

    14) Product Images from "Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl cis/trans Isomerases Identifies Three New PPIase Activities in Escherichia coli That Includes the DksA Transcription Factor"

    Article Title: Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl cis/trans Isomerases Identifies Three New PPIase Activities in Escherichia coli That Includes the DksA Transcription Factor

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21165843

    Catalysis of PPIase-dependent refolding of RNase T1. ( A ) The increase in fluorescence at 320 nm is depicted as a function of time of refolding of RNase T1 in the presence of DksA and PpiB. ( B ) Measurement of refolding of RNase T1 in the presence of wild-type DksA and different DksA mutants. ( C ) Refolding of RNase T1 catalyzed by the addition of Cmk as measured by the increase in fluorescence at 320 nm. Reaction when buffer alone is added is depicted in ( A , C ).
    Figure Legend Snippet: Catalysis of PPIase-dependent refolding of RNase T1. ( A ) The increase in fluorescence at 320 nm is depicted as a function of time of refolding of RNase T1 in the presence of DksA and PpiB. ( B ) Measurement of refolding of RNase T1 in the presence of wild-type DksA and different DksA mutants. ( C ) Refolding of RNase T1 catalyzed by the addition of Cmk as measured by the increase in fluorescence at 320 nm. Reaction when buffer alone is added is depicted in ( A , C ).

    Techniques Used: Fluorescence

    15) Product Images from "Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA"

    Article Title: Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp782

    RBM15 and OTT3 are required for general mRNA export. Human 293 cells were transfected with siRNAs targeting RBM15, OTT3 or non-targeting siRNA ( control ) and analyzed at day 2 or 4 posttransfection as indicated. ( A ) RT–qPCR detection of RBM15 and OTT3 transcripts at day 2. Expression levels were calculated from real-time PCR values ( C t ) using relative quantitation method and are plotted on the y -axis after normalization to those obtained in the cells transfected with the non-targeting siRNA control (normalized expression). Mean ( n =3) values are presented, and bars show one SEM. ( B ) Cells were transfected with the indicated siRNAs and, next day, with a plasmid expressing HA-OTT3 (1 μg). At day 2 or day 4 after siRNA transfection, cell pellets were boiled in Laemmli sample buffer, proteins separated on 10% SDS–PAGE and analyzed on western blots with antibodies to RBM15, HA, Ran, β-actin or SR proteins as indicated; or by Coomassie staining. ( C ) Cells at day 2 (left panel) or day 4 (right panel) posttransfection were separated into the C, N1 and N2 fractions, mRNA poly(A) tails were 3′-radiolabeled, cut off by RNase T1 digestion, separated by urea–PAGE and detected by phosphoimager. Positions of size markers (nt) are shown. ( D ) U snRNAs from the same fractions as in (D) were separated by urea–PAGE and detected on northern blots, and positions of the individual U snRNAs are indicated to the left. tRNA was detected on the same gels, by ethidium bromide staining prior to blotting (tRNA).
    Figure Legend Snippet: RBM15 and OTT3 are required for general mRNA export. Human 293 cells were transfected with siRNAs targeting RBM15, OTT3 or non-targeting siRNA ( control ) and analyzed at day 2 or 4 posttransfection as indicated. ( A ) RT–qPCR detection of RBM15 and OTT3 transcripts at day 2. Expression levels were calculated from real-time PCR values ( C t ) using relative quantitation method and are plotted on the y -axis after normalization to those obtained in the cells transfected with the non-targeting siRNA control (normalized expression). Mean ( n =3) values are presented, and bars show one SEM. ( B ) Cells were transfected with the indicated siRNAs and, next day, with a plasmid expressing HA-OTT3 (1 μg). At day 2 or day 4 after siRNA transfection, cell pellets were boiled in Laemmli sample buffer, proteins separated on 10% SDS–PAGE and analyzed on western blots with antibodies to RBM15, HA, Ran, β-actin or SR proteins as indicated; or by Coomassie staining. ( C ) Cells at day 2 (left panel) or day 4 (right panel) posttransfection were separated into the C, N1 and N2 fractions, mRNA poly(A) tails were 3′-radiolabeled, cut off by RNase T1 digestion, separated by urea–PAGE and detected by phosphoimager. Positions of size markers (nt) are shown. ( D ) U snRNAs from the same fractions as in (D) were separated by urea–PAGE and detected on northern blots, and positions of the individual U snRNAs are indicated to the left. tRNA was detected on the same gels, by ethidium bromide staining prior to blotting (tRNA).

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, Plasmid Preparation, SDS Page, Western Blot, Staining, Polyacrylamide Gel Electrophoresis, Northern Blot

    16) Product Images from "An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes"

    Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp732

    LC-MS analysis of RNaseT1 digests of yeast tRNA Phe−1 . ( a ) Base peak chromatogram of the RNase T1 digest of the yeast tRNA Phe−1 preparation (200 fmol). Chromatography was performed as described in the ‘Materials and Methods’ section. Sixteen major oligoribonucleotide peaks, indicated by arrows with peak numbers, are assigned to the fragments of yeast tRNA Phe−1 , tRNA Phe−2 , tRNA Lys−2 , or tRNA Tyr (see Table 1 ). ( b and c ) A typical MS spectrum of the RNase T1 digest of tRNA Phe−1 ; (b) [AUUUAm 2 G > p] 2− and (c) [ACmUGmAAyWAΨUm 5 CUG > p] 3− .
    Figure Legend Snippet: LC-MS analysis of RNaseT1 digests of yeast tRNA Phe−1 . ( a ) Base peak chromatogram of the RNase T1 digest of the yeast tRNA Phe−1 preparation (200 fmol). Chromatography was performed as described in the ‘Materials and Methods’ section. Sixteen major oligoribonucleotide peaks, indicated by arrows with peak numbers, are assigned to the fragments of yeast tRNA Phe−1 , tRNA Phe−2 , tRNA Lys−2 , or tRNA Tyr (see Table 1 ). ( b and c ) A typical MS spectrum of the RNase T1 digest of tRNA Phe−1 ; (b) [AUUUAm 2 G > p] 2− and (c) [ACmUGmAAyWAΨUm 5 CUG > p] 3− .

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Chromatography, Mass Spectrometry

    CID-MS/MS spectrum of [AUUUAm 2 G] 2− derived from RNase T1 digestion of yeast tRNA Phe−1 . The doubly charged ion with m / z = 966.6 was analyzed by CID. The nucleotide sequence was verified by manual interpretation of the a- and c-type (normal text) and w - and y -type ( italic text ) product ion series as indicated in the figure. The parent ion losing methyl guanine [P−B(mG) 2− ], the parent ion losing adenine [P−B(A) 2− ], the y5 ion losing methyl guanine [y5−B(mG) 2− ], the y5 ion losing adenine [y5−B(A) 2− ], y5 2− and c5 2− were doubly charged products. All other assigned signals were singly charged products, unless indicated otherwise. The asterisks indicate hydrated or dehydrated ions of a-, c-, w- or y-type products.
    Figure Legend Snippet: CID-MS/MS spectrum of [AUUUAm 2 G] 2− derived from RNase T1 digestion of yeast tRNA Phe−1 . The doubly charged ion with m / z = 966.6 was analyzed by CID. The nucleotide sequence was verified by manual interpretation of the a- and c-type (normal text) and w - and y -type ( italic text ) product ion series as indicated in the figure. The parent ion losing methyl guanine [P−B(mG) 2− ], the parent ion losing adenine [P−B(A) 2− ], the y5 ion losing methyl guanine [y5−B(mG) 2− ], the y5 ion losing adenine [y5−B(A) 2− ], y5 2− and c5 2− were doubly charged products. All other assigned signals were singly charged products, unless indicated otherwise. The asterisks indicate hydrated or dehydrated ions of a-, c-, w- or y-type products.

    Techniques Used: Mass Spectrometry, Derivative Assay, Sequencing

    Base peak chromatogram of the RNaseT1 digest of yeast U4 snRNA isolated from the Lsm3-associated RNP complex. The gel piece containing U4 snRNA was in-gel digested with RNaseT1 and subjected to the LC-MS analysis. Major oligoribonucleotide peaks assigned as RNase T1 fragments of yeast U4 snRNA are indicated by arrows with the corresponding sequence. Detailed data for MS/MS-based assignment of each fragment are given in Supplementary Table S3 .
    Figure Legend Snippet: Base peak chromatogram of the RNaseT1 digest of yeast U4 snRNA isolated from the Lsm3-associated RNP complex. The gel piece containing U4 snRNA was in-gel digested with RNaseT1 and subjected to the LC-MS analysis. Major oligoribonucleotide peaks assigned as RNase T1 fragments of yeast U4 snRNA are indicated by arrows with the corresponding sequence. Detailed data for MS/MS-based assignment of each fragment are given in Supplementary Table S3 .

    Techniques Used: Isolation, Liquid Chromatography with Mass Spectroscopy, Sequencing, Mass Spectrometry

    Related Articles

    Mutagenesis:

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile
    Article Snippet: .. Due to the mutation in the anticodon from G34 to U34, digestion with RNase T1 yields a different cleavage pattern for the wild-type and the mutant tRNA preparations ( and Supplementary Figure S2 ). .. In case of the wild-type tRNA, the anticodon loop is cleaved into two fragments, CCUGp and AUAAGp, while the mutant tRNA produces a mixture of three related 9-mers containing CCUUAUAAGp ( ).

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile
    Article Snippet: .. To remove the wild-type tRNA1 from the mixture of wild-type and mutant tRNA1 Ile , tRNA1 was treated with RNase T1 in the presence of Mg++ under mild conditions to inactivate specifically the wild-type tRNA1 by cleavage at position 34 ( ). .. RNase T1 treatment of tRNA1 produced a ‘nicked’ tRNA, which was inactive in both in vitro aminoacylation ( Supplementary Figure S5A ) and binding to ribosomes ( Supplementary Figure S5C ).

    Purification:

    Article Title: The complete chemical structure of Saccharomyces cerevisiae rRNA: partial pseudouridylation of U2345 in 25S rRNA by snoRNA snR9
    Article Snippet: .. Sodium cytidine-13 C9 5′-triphosphate (98 atom% 13 C) and sodium uridine-13 C9 5′-triphosphate (98 atom% 13 C) were purchased from Santa Cruz Biotechnology, Inc. Uracil-5-D (98 atom% 2 H) was obtained from C/D/N Isotopes, Inc. RNase T1 was purchased from Worthington and purified with reversed-phase LC before use. .. Triethylammonium acetate (TEAA, 1.0 M) buffer, pH 7.0, was purchased from Glen Research.

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile
    Article Snippet: .. First, purified tRNA1 and tRNA1 were digested completely with RNase T1 and the fragments produced were subjected to LC-MS/MS analysis. .. Due to the mutation in the anticodon from G34 to U34, digestion with RNase T1 yields a different cleavage pattern for the wild-type and the mutant tRNA preparations ( and Supplementary Figure S2 ).

    Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes
    Article Snippet: .. RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use. .. High-performance liquid chromatography grade methanol and acetonitrile were obtained from Kokusan Chemical Co. (Tokyo, Japan), and lysyl endopeptidase, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and acetic acid were obtained from Wako Pure Chemical Industries (Osaka, Japan).

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile
    Article Snippet: .. Mass spectral analysis of purified isoleucine tRNAs For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C. .. The digestion products were separated using a Thermo Surveyor HPLC system with a Waters XBridge C18 1.0 × 150 mm column at 40 μl/min with a gradient of 400 mM 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), 8.15 mM triethylamine (TEA), pH 7.0, and 400 mM HFIP, 8.15 mM TEA:methanol (50:50) (v:v ), pH 7.0.

    Produced:

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile
    Article Snippet: .. First, purified tRNA1 and tRNA1 were digested completely with RNase T1 and the fragments produced were subjected to LC-MS/MS analysis. .. Due to the mutation in the anticodon from G34 to U34, digestion with RNase T1 yields a different cleavage pattern for the wild-type and the mutant tRNA preparations ( and Supplementary Figure S2 ).

    other:

    Article Title: Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA
    Article Snippet: The poly(A)+ RNA from these fractions was radiolabeled at the 3′ ends of poly(A) tails and digested with RNase T1, leading to the complete hydrolysis of mRNA body while leaving the poly(A) tail intact.

    Sequencing:

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile
    Article Snippet: .. Mass spectral analysis of purified isoleucine tRNAs For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C. .. The digestion products were separated using a Thermo Surveyor HPLC system with a Waters XBridge C18 1.0 × 150 mm column at 40 μl/min with a gradient of 400 mM 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), 8.15 mM triethylamine (TEA), pH 7.0, and 400 mM HFIP, 8.15 mM TEA:methanol (50:50) (v:v ), pH 7.0.

    Liquid Chromatography:

    Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes
    Article Snippet: .. RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use. .. High-performance liquid chromatography grade methanol and acetonitrile were obtained from Kokusan Chemical Co. (Tokyo, Japan), and lysyl endopeptidase, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and acetic acid were obtained from Wako Pure Chemical Industries (Osaka, Japan).

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    Worthington Biochemical rnase t1
    Components of the S. pombe <t>RNase</t> MRP complex. (A) RNA component of S. pombe RNase MRP ( mrp1 ). RNA was separated from the purified RNase MRP with acid-phenol treatment and subjected to 8 M urea-7.5% PAGE (SYBR Gold staining). (B) Nucleotide sequence of S. pombe mrp1 RNA and the fragments used for the sequence analysis. Solid or dashed double-headed arrows show the fragments obtained by digestion with <t>RNase</t> T1 or MazF/PemK RNase, respectively. RNase T1 fragments were identified by Ariadne search program, and PemK/MazF fragments were identified by manual inspection of MS/MS spectra (see also Table S2 ). m 3 Gppp, trimethylguanosine cap. (C) Protein components of S. pombe RNase MRP. The RNase-MRP preparation affinity-purified using FEM3-tagged Rmp1 as bait (Rmp1-FEM3) was separated by SDS-PAGE and visualized with Coomassie Brilliant Blue staining. The proteins identified by LC-MS/MS are shown on the right (see also Table S3 and Figure S6 ). The IgG probably resulted from sloughing from the beads during the affinity purification.
    Rnase T1, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 93/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Components of the S. pombe RNase MRP complex. (A) RNA component of S. pombe RNase MRP ( mrp1 ). RNA was separated from the purified RNase MRP with acid-phenol treatment and subjected to 8 M urea-7.5% PAGE (SYBR Gold staining). (B) Nucleotide sequence of S. pombe mrp1 RNA and the fragments used for the sequence analysis. Solid or dashed double-headed arrows show the fragments obtained by digestion with RNase T1 or MazF/PemK RNase, respectively. RNase T1 fragments were identified by Ariadne search program, and PemK/MazF fragments were identified by manual inspection of MS/MS spectra (see also Table S2 ). m 3 Gppp, trimethylguanosine cap. (C) Protein components of S. pombe RNase MRP. The RNase-MRP preparation affinity-purified using FEM3-tagged Rmp1 as bait (Rmp1-FEM3) was separated by SDS-PAGE and visualized with Coomassie Brilliant Blue staining. The proteins identified by LC-MS/MS are shown on the right (see also Table S3 and Figure S6 ). The IgG probably resulted from sloughing from the beads during the affinity purification.

    Journal: PLoS ONE

    Article Title: RNase MRP Cleaves Pre-tRNASer-Met in the tRNA Maturation Pathway

    doi: 10.1371/journal.pone.0112488

    Figure Lengend Snippet: Components of the S. pombe RNase MRP complex. (A) RNA component of S. pombe RNase MRP ( mrp1 ). RNA was separated from the purified RNase MRP with acid-phenol treatment and subjected to 8 M urea-7.5% PAGE (SYBR Gold staining). (B) Nucleotide sequence of S. pombe mrp1 RNA and the fragments used for the sequence analysis. Solid or dashed double-headed arrows show the fragments obtained by digestion with RNase T1 or MazF/PemK RNase, respectively. RNase T1 fragments were identified by Ariadne search program, and PemK/MazF fragments were identified by manual inspection of MS/MS spectra (see also Table S2 ). m 3 Gppp, trimethylguanosine cap. (C) Protein components of S. pombe RNase MRP. The RNase-MRP preparation affinity-purified using FEM3-tagged Rmp1 as bait (Rmp1-FEM3) was separated by SDS-PAGE and visualized with Coomassie Brilliant Blue staining. The proteins identified by LC-MS/MS are shown on the right (see also Table S3 and Figure S6 ). The IgG probably resulted from sloughing from the beads during the affinity purification.

    Article Snippet: RNases for in-gel digestion, RNase T1 (Worthington), MazF (Takara Bio), and PemK were further purified before use .

    Techniques: Purification, Polyacrylamide Gel Electrophoresis, Staining, Sequencing, Mass Spectrometry, Affinity Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy

    Isolation of the catalytically active core of RNase MRP. (A) Urea-PAGE profiles of mrp1 RNA fragments produced by RNase A–mediated limited nucleolysis of RNase MRP (SYBR Gold staining). The S. pombe RNase MRP obtained from a 2-l culture of logarithmically growing cells was digested on FLAG M2 agarose beads at 4°C for 1 h with increasing amounts of RNase A. Lane 1, no RNase A; lane 2, 1 µg/ml; lane 3, 5 µg/ml; lane 4, 10 µg/ml. A portion (5%) of each reaction was loaded per lane. (B) A core of RNase MRP produced by RNase A–mediated limited nucleolysis cleaves pre-tRNA Ser-Met . RNase MRP (pulled down with tagged Rmp1 from JJ095 cells using FLAG M2 agarose) and the mock preparation (pulled down from SP6 cells) were incubated with (+) or without (−) RNase A. Each digested preparation (1 pmol each) was incubated with pre-tRNA Ser-Met (8 pmol) at 37°C for 30 min, and each reaction mixture was subjected to urea-PAGE (SYBR-Gold staining). Band 1, mrp1 RNA; 2, pre-tRNA Ser-Met ; 3, pre-tRNA Ser +trailer; 4, tRNA Met . (C) Double-reciprocal plot of the catalytic reaction of RNase A–mediated partial nucleolysis of RNase MRP. The reaction was performed for 30 min with synthetic pre-tRNA Ser-Met as a substrate under the conditions as in Figure 3B . The plot indicates K M of 0.974 µM and Vmax of 12.9 nM/min. (D) RNA fragments produced by RNase T1 digestion of Band 1 (indicated by solid lines) and Band 2 (broken lines) in (A). The fragments identified by LC-MS/MS are mapped on the mrp1 RNA sequence, where the conserved helices and strands of mrp1 [1] , [29] , [31] are shown in shaded boxes. (E) Nuclease-resistant regions mapped in the secondary structure of mrp1 . The map was according to the previous study [2] with modifications made by the assistance of CentroidHomFold ( http://www.ncrna.org/centroidhomfold ). Dashed-line boxes denote the two putative domains [2] . The nuclease-resistant region is shaded gray. The nucleotides with dotted bar are the consensus sequence ANAGNNA known as the mCR-IV motif [2] . (F) SDS-PAGE profiles of the protein components of RNase MRP before (−) and after (+) RNase A–mediated limited nucleolysis (Coomassie Brilliant Blue staining). The proteins assigned by LC-MS/MS are also shown. Note that the catalytic core of RNase MRP produced by partial nucleolysis contains 8 (underlined) of the 11 total subunits.

    Journal: PLoS ONE

    Article Title: RNase MRP Cleaves Pre-tRNASer-Met in the tRNA Maturation Pathway

    doi: 10.1371/journal.pone.0112488

    Figure Lengend Snippet: Isolation of the catalytically active core of RNase MRP. (A) Urea-PAGE profiles of mrp1 RNA fragments produced by RNase A–mediated limited nucleolysis of RNase MRP (SYBR Gold staining). The S. pombe RNase MRP obtained from a 2-l culture of logarithmically growing cells was digested on FLAG M2 agarose beads at 4°C for 1 h with increasing amounts of RNase A. Lane 1, no RNase A; lane 2, 1 µg/ml; lane 3, 5 µg/ml; lane 4, 10 µg/ml. A portion (5%) of each reaction was loaded per lane. (B) A core of RNase MRP produced by RNase A–mediated limited nucleolysis cleaves pre-tRNA Ser-Met . RNase MRP (pulled down with tagged Rmp1 from JJ095 cells using FLAG M2 agarose) and the mock preparation (pulled down from SP6 cells) were incubated with (+) or without (−) RNase A. Each digested preparation (1 pmol each) was incubated with pre-tRNA Ser-Met (8 pmol) at 37°C for 30 min, and each reaction mixture was subjected to urea-PAGE (SYBR-Gold staining). Band 1, mrp1 RNA; 2, pre-tRNA Ser-Met ; 3, pre-tRNA Ser +trailer; 4, tRNA Met . (C) Double-reciprocal plot of the catalytic reaction of RNase A–mediated partial nucleolysis of RNase MRP. The reaction was performed for 30 min with synthetic pre-tRNA Ser-Met as a substrate under the conditions as in Figure 3B . The plot indicates K M of 0.974 µM and Vmax of 12.9 nM/min. (D) RNA fragments produced by RNase T1 digestion of Band 1 (indicated by solid lines) and Band 2 (broken lines) in (A). The fragments identified by LC-MS/MS are mapped on the mrp1 RNA sequence, where the conserved helices and strands of mrp1 [1] , [29] , [31] are shown in shaded boxes. (E) Nuclease-resistant regions mapped in the secondary structure of mrp1 . The map was according to the previous study [2] with modifications made by the assistance of CentroidHomFold ( http://www.ncrna.org/centroidhomfold ). Dashed-line boxes denote the two putative domains [2] . The nuclease-resistant region is shaded gray. The nucleotides with dotted bar are the consensus sequence ANAGNNA known as the mCR-IV motif [2] . (F) SDS-PAGE profiles of the protein components of RNase MRP before (−) and after (+) RNase A–mediated limited nucleolysis (Coomassie Brilliant Blue staining). The proteins assigned by LC-MS/MS are also shown. Note that the catalytic core of RNase MRP produced by partial nucleolysis contains 8 (underlined) of the 11 total subunits.

    Article Snippet: RNases for in-gel digestion, RNase T1 (Worthington), MazF (Takara Bio), and PemK were further purified before use .

    Techniques: Isolation, Polyacrylamide Gel Electrophoresis, Produced, Staining, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, SDS Page

    Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.

    Journal: Nucleic Acids Research

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    doi: 10.1093/nar/gkt1009

    Figure Lengend Snippet: Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.

    Article Snippet: Mass spectral analysis of purified isoleucine tRNAs For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C.

    Techniques: Binding Assay, Purification, Mutagenesis, Isolation, Concentration Assay

    Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.

    Journal: Nucleic Acids Research

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    doi: 10.1093/nar/gkt1009

    Figure Lengend Snippet: Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.

    Article Snippet: Mass spectral analysis of purified isoleucine tRNAs For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C.

    Techniques: Purification, Mutagenesis, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Clear Native PAGE, Staining, Northern Blot, Hybridization

    A: The total ion chromatogram (TIC) of RNase T1 digested E. coli tRNA Gln and the accompanying extracted ion chromatogram (XIC) of m/z 1004, which corresponds to the expected m/z for the fragment with the sequence U[Um]U[cmnm 5 s 2 U]UGp. B: The MS spectrum of the eluent at 28.2 min. depicting the m/z 1004.25, consistent with the sequence U[Um]U[cmnm 5 s 2 U]UGp. C: The peak in B was selected for MS/MS analysis. The resulting fragmentation is consistent with U[Um]U[cmnm 5 s 2 U]UGp with all identified c- and y-type product ions labeled.

    Journal: Journal of molecular biology

    Article Title: Structural and mechanistic basis for enhanced translational efficiency by 2-thiouridine at the tRNA anticodon wobble position

    doi: 10.1016/j.jmb.2013.05.018

    Figure Lengend Snippet: A: The total ion chromatogram (TIC) of RNase T1 digested E. coli tRNA Gln and the accompanying extracted ion chromatogram (XIC) of m/z 1004, which corresponds to the expected m/z for the fragment with the sequence U[Um]U[cmnm 5 s 2 U]UGp. B: The MS spectrum of the eluent at 28.2 min. depicting the m/z 1004.25, consistent with the sequence U[Um]U[cmnm 5 s 2 U]UGp. C: The peak in B was selected for MS/MS analysis. The resulting fragmentation is consistent with U[Um]U[cmnm 5 s 2 U]UGp with all identified c- and y-type product ions labeled.

    Article Snippet: For sequence analysis, purified tRNA1 Gln from E. coli was digested with 50 U/μg of RNase T1 (Worthington Biochemical Corporation) in 20 mM ammonium acetate for 2 h at 37 °C.

    Techniques: Sequencing, Mass Spectrometry, Labeling