trar  (Worthington Biochemical)


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    • 91
    Name:
    Deoxyribonuclease I
    Description:
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    Catalog Number:
    ls002004
    Price:
    33
    Size:
    5 mg
    Source:
    Bovine Pancreas
    Cas Number:
    9003.98.9
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    Structured Review

    Worthington Biochemical trar
    Models for <t>TraR</t> and the TraR–RNAP complex. ( A ) RaptorX-derived model for TraR. N- and C-termini are indicated, the four cysteine residues (C4) are tan spheres, and residues D3, D6, A8, I20, and E66 are in blue stick form. ( B ) TraR (blue) and <t>DksA</t> (green) were positioned manually based on alignment of TraR D3, D6, and A8 with DksA D71, D74, and A76, a portion of the TraR N-terminal α-helix, and the DksA α-helix 2 in its coiled-coil. Cysteines (C4) are yellow or tan spheres. ( C ) Model for TraR binding to E. coli ). The square corresponds to the area of the complex shown in expanded form in D and E . TraR is dark blue; the RNAP β-subunit is cyan, β′ is pink, the β′ secondary channel rim is yellow, and ω is pale blue. β′ Residues N680, K681A, and E677 are shown as orange spheres. ppGpp at site 1 is shown in red. TraR residues D3, D6, I44, A47, R48, I51, A47, and E66 are shown as blue spheres. ( D ) Enlarged view of TraR bound to RNAP secondary channel, as in C . I20 is in stick form. BH, bridge helix. ( E .
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    https://www.bioz.com/result/trar/product/Worthington Biochemical
    Average 91 stars, based on 640 article reviews
    Price from $9.99 to $1999.99
    trar - by Bioz Stars, 2020-05
    91/100 stars

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    Images

    1) Product Images from "TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp"

    Article Title: TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1704105114

    Models for TraR and the TraR–RNAP complex. ( A ) RaptorX-derived model for TraR. N- and C-termini are indicated, the four cysteine residues (C4) are tan spheres, and residues D3, D6, A8, I20, and E66 are in blue stick form. ( B ) TraR (blue) and DksA (green) were positioned manually based on alignment of TraR D3, D6, and A8 with DksA D71, D74, and A76, a portion of the TraR N-terminal α-helix, and the DksA α-helix 2 in its coiled-coil. Cysteines (C4) are yellow or tan spheres. ( C ) Model for TraR binding to E. coli ). The square corresponds to the area of the complex shown in expanded form in D and E . TraR is dark blue; the RNAP β-subunit is cyan, β′ is pink, the β′ secondary channel rim is yellow, and ω is pale blue. β′ Residues N680, K681A, and E677 are shown as orange spheres. ppGpp at site 1 is shown in red. TraR residues D3, D6, I44, A47, R48, I51, A47, and E66 are shown as blue spheres. ( D ) Enlarged view of TraR bound to RNAP secondary channel, as in C . I20 is in stick form. BH, bridge helix. ( E .
    Figure Legend Snippet: Models for TraR and the TraR–RNAP complex. ( A ) RaptorX-derived model for TraR. N- and C-termini are indicated, the four cysteine residues (C4) are tan spheres, and residues D3, D6, A8, I20, and E66 are in blue stick form. ( B ) TraR (blue) and DksA (green) were positioned manually based on alignment of TraR D3, D6, and A8 with DksA D71, D74, and A76, a portion of the TraR N-terminal α-helix, and the DksA α-helix 2 in its coiled-coil. Cysteines (C4) are yellow or tan spheres. ( C ) Model for TraR binding to E. coli ). The square corresponds to the area of the complex shown in expanded form in D and E . TraR is dark blue; the RNAP β-subunit is cyan, β′ is pink, the β′ secondary channel rim is yellow, and ω is pale blue. β′ Residues N680, K681A, and E677 are shown as orange spheres. ppGpp at site 1 is shown in red. TraR residues D3, D6, I44, A47, R48, I51, A47, and E66 are shown as blue spheres. ( D ) Enlarged view of TraR bound to RNAP secondary channel, as in C . I20 is in stick form. BH, bridge helix. ( E .

    Techniques Used: Derivative Assay, Binding Assay

    ( A ) Activation of p hisG , p livJ , or RNA-1 by TraR (0–500 nM). ( B ) Activation shown reflects maximum activation observed. Transcription of iraP P1 is activated to a different extent by TraR vs. DksA/ppGpp. Values plotted with TraR (1 µM), DksA (1 µM), or DksA (2 µM) and ppGpp (50 µM) together, relative to transcription without factors (set to 1).
    Figure Legend Snippet: ( A ) Activation of p hisG , p livJ , or RNA-1 by TraR (0–500 nM). ( B ) Activation shown reflects maximum activation observed. Transcription of iraP P1 is activated to a different extent by TraR vs. DksA/ppGpp. Values plotted with TraR (1 µM), DksA (1 µM), or DksA (2 µM) and ppGpp (50 µM) together, relative to transcription without factors (set to 1).

    Techniques Used: Activation Assay

    A half-DksA variant, similar in length to WT TraR, does not complement a ∆ dksA mutant. ( A ) Representative Western blot from cell lysates made from a ∆ dksA strain carrying either the WT gene or the half - dksA gene fused to an IPTG-inducible promoter. Cells were harvested 1, 2, or 3 h after induction with 0.5 mM IPTG. One-microgram of cell lysate was loaded in each lane, and purified DksA-HMK was loaded in lane 1 for comparison. Somewhat lower amounts of the half-DksA variant were observed than of WT DksA, which could be attributable to lower stability of the half-DksA variant or to reduced ability of the DksA antibody to recognize the half-DksA peptide. ( B and C ) Growth on plates containing defined medium without amino acids and ( B ) 0.1 mM IPTG or ( C ) 1 mM IPTG. Sector 1: empty vector control. Sector 2: plasmid containing half - dksA gene. Sector 3: plasmid containing the full-length traR gene. Sector 4: plasmid containing the full-length dksA gene. Even if the half-DksA concentration was lower than the WT DksA concentration, it is likely that the half-DksA would have still resulted in at least partial complementation, because we showed previously that even 50% of the WT concentration supplied from a plasmid was sufficient to complement a ∆ dksA ).
    Figure Legend Snippet: A half-DksA variant, similar in length to WT TraR, does not complement a ∆ dksA mutant. ( A ) Representative Western blot from cell lysates made from a ∆ dksA strain carrying either the WT gene or the half - dksA gene fused to an IPTG-inducible promoter. Cells were harvested 1, 2, or 3 h after induction with 0.5 mM IPTG. One-microgram of cell lysate was loaded in each lane, and purified DksA-HMK was loaded in lane 1 for comparison. Somewhat lower amounts of the half-DksA variant were observed than of WT DksA, which could be attributable to lower stability of the half-DksA variant or to reduced ability of the DksA antibody to recognize the half-DksA peptide. ( B and C ) Growth on plates containing defined medium without amino acids and ( B ) 0.1 mM IPTG or ( C ) 1 mM IPTG. Sector 1: empty vector control. Sector 2: plasmid containing half - dksA gene. Sector 3: plasmid containing the full-length traR gene. Sector 4: plasmid containing the full-length dksA gene. Even if the half-DksA concentration was lower than the WT DksA concentration, it is likely that the half-DksA would have still resulted in at least partial complementation, because we showed previously that even 50% of the WT concentration supplied from a plasmid was sufficient to complement a ∆ dksA ).

    Techniques Used: Variant Assay, Mutagenesis, Western Blot, Purification, Plasmid Preparation, Concentration Assay

    TraR is more active than DksA for inhibition of transcription but has a similar affinity for RNAP. ( A ) Multiround in vitro transcription of rrnB P1 or lacUV 5 at a range of concentrations of TraR (wedge indicates 1 nM to 1 µM for rrnB P1 or 1 nM to 2 µM for lacUV5 ) or of DksA (wedge indicates 4 nM to 8 µM). Plasmid templates also contained the RNA-1 promoter. ( B ) Quantification of transcripts from experiments like those in A plotted relative to values in the absence of TraR or DksA. The IC 50 for inhibition by TraR was ∼50 nM and for DksA ∼1.3 µM [averages with SDs from at least three independent experiments ( n = 3)]. ( C ) Cross-linking with β′ R933-Bpa RNAP, β′ Q929-Bpa RNAP, or β′ R1148-Bpa RNAP with 32 P-TraR or 32 P-DksA. The portion of a representative 4–12% SDS gel containing the cross-linked β′-DksA or β′-TraR products is shown. ( D ) Unlabeled DksA or TraR competes similarly for binding of 32 P-labeled HMK-DksA to RNAP. Unlabeled DksA or TraR (0–16 µM) was added to 1 µM 32 P-DksA and 0.1 µM core RNAP before Fe 2+ -mediated cleavage of DksA. Fraction of 32 P-DksA cleaved was normalized to that in the absence of competitor. Next, 1 µM unlabeled DksA or 0.6 µM unlabeled TraR reduced cleavage of 1 µM 32 P-DksA by ∼50% ( n = 3). ( E ) Representative gel showing DNase I footprints of RNAP bound to the rrnB P1 promoter, 3′ end-labeled on the template strand, with or without TraR or DksA. DNase I digested fragment without RNAP or added factors (lanes 1 and 2), with RNAP alone (lanes 3 and 4), with RNAP + 5 µM DksA (lanes 5 and 6), or with RNAP and 5 µM TraR (lanes 7 and 8). Undigested fragment (lane 9). A+G sequence ladder is on the Left . Traces of gel lanes showing extent of protection are on the Right . Colored dots indicate the downstream boundary of DNase I protection without (green dot; ∼+12), or with (red or blue dots; ∼+1) DksA or TraR. The upstream boundary of protection in lanes 3–8 is ∼−59 ( n = 3). ( F ) TraR and DksA alter the lifetime of rrnB P1(dis) promoter complexes in vitro. RNAP–promoter complexes were preformed with TraR (15 nM) or DksA (15 nM or 500 nM), or without factors, and the fraction remaining at the indicated times after heparin addition was determined by transcription. Half-lives of rrnB P1(dis) complexes: no added factor, 18 min; 15 nM TraR, 3 min; 15 nM DksA, 18 min; 500 nM DksA, 6 min. Error bars indicate the range from two independent experiments ( n = 2).
    Figure Legend Snippet: TraR is more active than DksA for inhibition of transcription but has a similar affinity for RNAP. ( A ) Multiround in vitro transcription of rrnB P1 or lacUV 5 at a range of concentrations of TraR (wedge indicates 1 nM to 1 µM for rrnB P1 or 1 nM to 2 µM for lacUV5 ) or of DksA (wedge indicates 4 nM to 8 µM). Plasmid templates also contained the RNA-1 promoter. ( B ) Quantification of transcripts from experiments like those in A plotted relative to values in the absence of TraR or DksA. The IC 50 for inhibition by TraR was ∼50 nM and for DksA ∼1.3 µM [averages with SDs from at least three independent experiments ( n = 3)]. ( C ) Cross-linking with β′ R933-Bpa RNAP, β′ Q929-Bpa RNAP, or β′ R1148-Bpa RNAP with 32 P-TraR or 32 P-DksA. The portion of a representative 4–12% SDS gel containing the cross-linked β′-DksA or β′-TraR products is shown. ( D ) Unlabeled DksA or TraR competes similarly for binding of 32 P-labeled HMK-DksA to RNAP. Unlabeled DksA or TraR (0–16 µM) was added to 1 µM 32 P-DksA and 0.1 µM core RNAP before Fe 2+ -mediated cleavage of DksA. Fraction of 32 P-DksA cleaved was normalized to that in the absence of competitor. Next, 1 µM unlabeled DksA or 0.6 µM unlabeled TraR reduced cleavage of 1 µM 32 P-DksA by ∼50% ( n = 3). ( E ) Representative gel showing DNase I footprints of RNAP bound to the rrnB P1 promoter, 3′ end-labeled on the template strand, with or without TraR or DksA. DNase I digested fragment without RNAP or added factors (lanes 1 and 2), with RNAP alone (lanes 3 and 4), with RNAP + 5 µM DksA (lanes 5 and 6), or with RNAP and 5 µM TraR (lanes 7 and 8). Undigested fragment (lane 9). A+G sequence ladder is on the Left . Traces of gel lanes showing extent of protection are on the Right . Colored dots indicate the downstream boundary of DNase I protection without (green dot; ∼+12), or with (red or blue dots; ∼+1) DksA or TraR. The upstream boundary of protection in lanes 3–8 is ∼−59 ( n = 3). ( F ) TraR and DksA alter the lifetime of rrnB P1(dis) promoter complexes in vitro. RNAP–promoter complexes were preformed with TraR (15 nM) or DksA (15 nM or 500 nM), or without factors, and the fraction remaining at the indicated times after heparin addition was determined by transcription. Half-lives of rrnB P1(dis) complexes: no added factor, 18 min; 15 nM TraR, 3 min; 15 nM DksA, 18 min; 500 nM DksA, 6 min. Error bars indicate the range from two independent experiments ( n = 2).

    Techniques Used: Inhibition, In Vitro, Plasmid Preparation, SDS-Gel, Binding Assay, Labeling, Sequencing

    2) Product Images from "TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp"

    Article Title: TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1704105114

    Models for TraR and the TraR–RNAP complex. ( A ) RaptorX-derived model for TraR. N- and C-termini are indicated, the four cysteine residues (C4) are tan spheres, and residues D3, D6, A8, I20, and E66 are in blue stick form. ( B ) TraR (blue) and DksA (green) were positioned manually based on alignment of TraR D3, D6, and A8 with DksA D71, D74, and A76, a portion of the TraR N-terminal α-helix, and the DksA α-helix 2 in its coiled-coil. Cysteines (C4) are yellow or tan spheres. ( C ) Model for TraR binding to E. coli ). The square corresponds to the area of the complex shown in expanded form in D and E . TraR is dark blue; the RNAP β-subunit is cyan, β′ is pink, the β′ secondary channel rim is yellow, and ω is pale blue. β′ Residues N680, K681A, and E677 are shown as orange spheres. ppGpp at site 1 is shown in red. TraR residues D3, D6, I44, A47, R48, I51, A47, and E66 are shown as blue spheres. ( D ) Enlarged view of TraR bound to RNAP secondary channel, as in C . I20 is in stick form. BH, bridge helix. ( E .
    Figure Legend Snippet: Models for TraR and the TraR–RNAP complex. ( A ) RaptorX-derived model for TraR. N- and C-termini are indicated, the four cysteine residues (C4) are tan spheres, and residues D3, D6, A8, I20, and E66 are in blue stick form. ( B ) TraR (blue) and DksA (green) were positioned manually based on alignment of TraR D3, D6, and A8 with DksA D71, D74, and A76, a portion of the TraR N-terminal α-helix, and the DksA α-helix 2 in its coiled-coil. Cysteines (C4) are yellow or tan spheres. ( C ) Model for TraR binding to E. coli ). The square corresponds to the area of the complex shown in expanded form in D and E . TraR is dark blue; the RNAP β-subunit is cyan, β′ is pink, the β′ secondary channel rim is yellow, and ω is pale blue. β′ Residues N680, K681A, and E677 are shown as orange spheres. ppGpp at site 1 is shown in red. TraR residues D3, D6, I44, A47, R48, I51, A47, and E66 are shown as blue spheres. ( D ) Enlarged view of TraR bound to RNAP secondary channel, as in C . I20 is in stick form. BH, bridge helix. ( E .

    Techniques Used: Derivative Assay, Binding Assay

    ( A ) Activation of p hisG , p livJ , or RNA-1 by TraR (0–500 nM). ( B ) Activation shown reflects maximum activation observed. Transcription of iraP P1 is activated to a different extent by TraR vs. DksA/ppGpp. Values plotted with TraR (1 µM), DksA (1 µM), or DksA (2 µM) and ppGpp (50 µM) together, relative to transcription without factors (set to 1).
    Figure Legend Snippet: ( A ) Activation of p hisG , p livJ , or RNA-1 by TraR (0–500 nM). ( B ) Activation shown reflects maximum activation observed. Transcription of iraP P1 is activated to a different extent by TraR vs. DksA/ppGpp. Values plotted with TraR (1 µM), DksA (1 µM), or DksA (2 µM) and ppGpp (50 µM) together, relative to transcription without factors (set to 1).

    Techniques Used: Activation Assay

    A half-DksA variant, similar in length to WT TraR, does not complement a ∆ dksA mutant. ( A ) Representative Western blot from cell lysates made from a ∆ dksA strain carrying either the WT gene or the half - dksA gene fused to an IPTG-inducible promoter. Cells were harvested 1, 2, or 3 h after induction with 0.5 mM IPTG. One-microgram of cell lysate was loaded in each lane, and purified DksA-HMK was loaded in lane 1 for comparison. Somewhat lower amounts of the half-DksA variant were observed than of WT DksA, which could be attributable to lower stability of the half-DksA variant or to reduced ability of the DksA antibody to recognize the half-DksA peptide. ( B and C ) Growth on plates containing defined medium without amino acids and ( B ) 0.1 mM IPTG or ( C ) 1 mM IPTG. Sector 1: empty vector control. Sector 2: plasmid containing half - dksA gene. Sector 3: plasmid containing the full-length traR gene. Sector 4: plasmid containing the full-length dksA gene. Even if the half-DksA concentration was lower than the WT DksA concentration, it is likely that the half-DksA would have still resulted in at least partial complementation, because we showed previously that even 50% of the WT concentration supplied from a plasmid was sufficient to complement a ∆ dksA ).
    Figure Legend Snippet: A half-DksA variant, similar in length to WT TraR, does not complement a ∆ dksA mutant. ( A ) Representative Western blot from cell lysates made from a ∆ dksA strain carrying either the WT gene or the half - dksA gene fused to an IPTG-inducible promoter. Cells were harvested 1, 2, or 3 h after induction with 0.5 mM IPTG. One-microgram of cell lysate was loaded in each lane, and purified DksA-HMK was loaded in lane 1 for comparison. Somewhat lower amounts of the half-DksA variant were observed than of WT DksA, which could be attributable to lower stability of the half-DksA variant or to reduced ability of the DksA antibody to recognize the half-DksA peptide. ( B and C ) Growth on plates containing defined medium without amino acids and ( B ) 0.1 mM IPTG or ( C ) 1 mM IPTG. Sector 1: empty vector control. Sector 2: plasmid containing half - dksA gene. Sector 3: plasmid containing the full-length traR gene. Sector 4: plasmid containing the full-length dksA gene. Even if the half-DksA concentration was lower than the WT DksA concentration, it is likely that the half-DksA would have still resulted in at least partial complementation, because we showed previously that even 50% of the WT concentration supplied from a plasmid was sufficient to complement a ∆ dksA ).

    Techniques Used: Variant Assay, Mutagenesis, Western Blot, Purification, Plasmid Preparation, Concentration Assay

    TraR is more active than DksA for inhibition of transcription but has a similar affinity for RNAP. ( A ) Multiround in vitro transcription of rrnB P1 or lacUV 5 at a range of concentrations of TraR (wedge indicates 1 nM to 1 µM for rrnB P1 or 1 nM to 2 µM for lacUV5 ) or of DksA (wedge indicates 4 nM to 8 µM). Plasmid templates also contained the RNA-1 promoter. ( B ) Quantification of transcripts from experiments like those in A plotted relative to values in the absence of TraR or DksA. The IC 50 for inhibition by TraR was ∼50 nM and for DksA ∼1.3 µM [averages with SDs from at least three independent experiments ( n = 3)]. ( C ) Cross-linking with β′ R933-Bpa RNAP, β′ Q929-Bpa RNAP, or β′ R1148-Bpa RNAP with 32 P-TraR or 32 P-DksA. The portion of a representative 4–12% SDS gel containing the cross-linked β′-DksA or β′-TraR products is shown. ( D ) Unlabeled DksA or TraR competes similarly for binding of 32 P-labeled HMK-DksA to RNAP. Unlabeled DksA or TraR (0–16 µM) was added to 1 µM 32 P-DksA and 0.1 µM core RNAP before Fe 2+ -mediated cleavage of DksA. Fraction of 32 P-DksA cleaved was normalized to that in the absence of competitor. Next, 1 µM unlabeled DksA or 0.6 µM unlabeled TraR reduced cleavage of 1 µM 32 P-DksA by ∼50% ( n = 3). ( E ) Representative gel showing DNase I footprints of RNAP bound to the rrnB P1 promoter, 3′ end-labeled on the template strand, with or without TraR or DksA. DNase I digested fragment without RNAP or added factors (lanes 1 and 2), with RNAP alone (lanes 3 and 4), with RNAP + 5 µM DksA (lanes 5 and 6), or with RNAP and 5 µM TraR (lanes 7 and 8). Undigested fragment (lane 9). A+G sequence ladder is on the Left . Traces of gel lanes showing extent of protection are on the Right . Colored dots indicate the downstream boundary of DNase I protection without (green dot; ∼+12), or with (red or blue dots; ∼+1) DksA or TraR. The upstream boundary of protection in lanes 3–8 is ∼−59 ( n = 3). ( F ) TraR and DksA alter the lifetime of rrnB P1(dis) promoter complexes in vitro. RNAP–promoter complexes were preformed with TraR (15 nM) or DksA (15 nM or 500 nM), or without factors, and the fraction remaining at the indicated times after heparin addition was determined by transcription. Half-lives of rrnB P1(dis) complexes: no added factor, 18 min; 15 nM TraR, 3 min; 15 nM DksA, 18 min; 500 nM DksA, 6 min. Error bars indicate the range from two independent experiments ( n = 2).
    Figure Legend Snippet: TraR is more active than DksA for inhibition of transcription but has a similar affinity for RNAP. ( A ) Multiround in vitro transcription of rrnB P1 or lacUV 5 at a range of concentrations of TraR (wedge indicates 1 nM to 1 µM for rrnB P1 or 1 nM to 2 µM for lacUV5 ) or of DksA (wedge indicates 4 nM to 8 µM). Plasmid templates also contained the RNA-1 promoter. ( B ) Quantification of transcripts from experiments like those in A plotted relative to values in the absence of TraR or DksA. The IC 50 for inhibition by TraR was ∼50 nM and for DksA ∼1.3 µM [averages with SDs from at least three independent experiments ( n = 3)]. ( C ) Cross-linking with β′ R933-Bpa RNAP, β′ Q929-Bpa RNAP, or β′ R1148-Bpa RNAP with 32 P-TraR or 32 P-DksA. The portion of a representative 4–12% SDS gel containing the cross-linked β′-DksA or β′-TraR products is shown. ( D ) Unlabeled DksA or TraR competes similarly for binding of 32 P-labeled HMK-DksA to RNAP. Unlabeled DksA or TraR (0–16 µM) was added to 1 µM 32 P-DksA and 0.1 µM core RNAP before Fe 2+ -mediated cleavage of DksA. Fraction of 32 P-DksA cleaved was normalized to that in the absence of competitor. Next, 1 µM unlabeled DksA or 0.6 µM unlabeled TraR reduced cleavage of 1 µM 32 P-DksA by ∼50% ( n = 3). ( E ) Representative gel showing DNase I footprints of RNAP bound to the rrnB P1 promoter, 3′ end-labeled on the template strand, with or without TraR or DksA. DNase I digested fragment without RNAP or added factors (lanes 1 and 2), with RNAP alone (lanes 3 and 4), with RNAP + 5 µM DksA (lanes 5 and 6), or with RNAP and 5 µM TraR (lanes 7 and 8). Undigested fragment (lane 9). A+G sequence ladder is on the Left . Traces of gel lanes showing extent of protection are on the Right . Colored dots indicate the downstream boundary of DNase I protection without (green dot; ∼+12), or with (red or blue dots; ∼+1) DksA or TraR. The upstream boundary of protection in lanes 3–8 is ∼−59 ( n = 3). ( F ) TraR and DksA alter the lifetime of rrnB P1(dis) promoter complexes in vitro. RNAP–promoter complexes were preformed with TraR (15 nM) or DksA (15 nM or 500 nM), or without factors, and the fraction remaining at the indicated times after heparin addition was determined by transcription. Half-lives of rrnB P1(dis) complexes: no added factor, 18 min; 15 nM TraR, 3 min; 15 nM DksA, 18 min; 500 nM DksA, 6 min. Error bars indicate the range from two independent experiments ( n = 2).

    Techniques Used: Inhibition, In Vitro, Plasmid Preparation, SDS-Gel, Binding Assay, Labeling, Sequencing

    3) Product Images from "A mutant RNA polymerase that forms unusual open promoter complexes"

    Article Title: A mutant RNA polymerase that forms unusual open promoter complexes

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    The mutant RNAP promoter complexes are stable at low temperatures. ( A ) DNase I footprinting and KMnO 4 probing of promoter complexes at different temperatures (bottom strand). Promoter complexes were formed at the indicated temperatures and followed by DNase I and KMnO 4 treatment. ( B ) KMnO 4 probing of promoter complexes formed at −20°C in 50% glycerol to prevent freezing. The 37°C controls were conducted in the presence (lanes 4 and 6) or the absence (lanes 3 and 5) of 50% glycerol in the reaction buffer. The results indicate that open complex formation and KMnO 4 sensitivity were not affected by the glycerol. See Materials and Methods for experimental details. ( C ) Quantitative KMnO 4 probing of promoter complexes formed at different temperatures. Promoter complex formation reactions were set up on ice or 37°C, brought to the assay temperature, incubated for 15 min, and probed with KMnO 4 . The reaction products were separated by denaturing PAGE ( Top ) and quantified as described in Materials and Methods ( Bottom ). KMnO 4 probing was performed at 0 (lanes 1, 12, 13, and 24); 5 (lanes 2, 11, 14, and 23); 10 (lanes 3, 10, 15, and 22); 15 (lanes 4, 9, 16, and 21); 23 (lanes 5, 8, 17, and 20); and 37°C (lanes 6, 7, 18, and 19). The upward vertical arrows indicate an up-temperature shift from 0°C to the assay temperature; downward arrows indicate a down-temperature shift from 37°C to the assay temperature.
    Figure Legend Snippet: The mutant RNAP promoter complexes are stable at low temperatures. ( A ) DNase I footprinting and KMnO 4 probing of promoter complexes at different temperatures (bottom strand). Promoter complexes were formed at the indicated temperatures and followed by DNase I and KMnO 4 treatment. ( B ) KMnO 4 probing of promoter complexes formed at −20°C in 50% glycerol to prevent freezing. The 37°C controls were conducted in the presence (lanes 4 and 6) or the absence (lanes 3 and 5) of 50% glycerol in the reaction buffer. The results indicate that open complex formation and KMnO 4 sensitivity were not affected by the glycerol. See Materials and Methods for experimental details. ( C ) Quantitative KMnO 4 probing of promoter complexes formed at different temperatures. Promoter complex formation reactions were set up on ice or 37°C, brought to the assay temperature, incubated for 15 min, and probed with KMnO 4 . The reaction products were separated by denaturing PAGE ( Top ) and quantified as described in Materials and Methods ( Bottom ). KMnO 4 probing was performed at 0 (lanes 1, 12, 13, and 24); 5 (lanes 2, 11, 14, and 23); 10 (lanes 3, 10, 15, and 22); 15 (lanes 4, 9, 16, and 21); 23 (lanes 5, 8, 17, and 20); and 37°C (lanes 6, 7, 18, and 19). The upward vertical arrows indicate an up-temperature shift from 0°C to the assay temperature; downward arrows indicate a down-temperature shift from 37°C to the assay temperature.

    Techniques Used: Mutagenesis, Footprinting, Incubation, Polyacrylamide Gel Electrophoresis

    The mutant RNAP forms stable promoter complexes that lack protein–DNA contacts downstream of the transcription initiation start point. ( A ) 100 nM of a 106-bp DNA fragment containing the T7 A2 promoter (−84 to +32) 32 P-end-labeled on the bottom strand was combined with 200 nM wt or mutant RNAP in 20-μl reactions containing 40 mM Tris⋅HCl (pH 7.9), 40 mM KCl, and 10 mM MgCl 2 . Reactions were preincubated for 15 min at 37°C and footprinted with DNase I or probed with KMnO 4 . Reaction products were resolved on a 6% sequencing gel and visualized by autoradiography. ( B ) Summary of the RNAP–DNA footprinting experiments on the top and bottom strands of the T7 A2 promoter. Reactive thymines are indicated by arrows. DNase I-protected and KMnO 4 -sensitive sites found in the mutant and the wt promoter complexes are shown in black. DNase I-protected and KMnO 4 -sensitive sites specific for the wt complex are shown in gray. ( C ) Heparin resistance of RNAP-T7 A2 promoter complexes. Promoter complexes were formed as described above, and heparin was added to the final concentration 200 μg/ml (time 0), and incubation at 37°C was continued. At various time points, reaction aliquots were withdrawn, probed with KMnO 4 , resolved by denaturing PAGE, and quantified by using a PhosphorImager (Molecular Dynamics). The mean values of three independent measurements are given. The error bars represent the SD of the measurements. The normalized opening (%) value was calculated as described in Materials and Methods . ( D ) Reactions were set up as described above in a buffer containing the indicated concentrations of NaCl. Reactions were incubated for 15 min at 37°C and analyzed as in C .
    Figure Legend Snippet: The mutant RNAP forms stable promoter complexes that lack protein–DNA contacts downstream of the transcription initiation start point. ( A ) 100 nM of a 106-bp DNA fragment containing the T7 A2 promoter (−84 to +32) 32 P-end-labeled on the bottom strand was combined with 200 nM wt or mutant RNAP in 20-μl reactions containing 40 mM Tris⋅HCl (pH 7.9), 40 mM KCl, and 10 mM MgCl 2 . Reactions were preincubated for 15 min at 37°C and footprinted with DNase I or probed with KMnO 4 . Reaction products were resolved on a 6% sequencing gel and visualized by autoradiography. ( B ) Summary of the RNAP–DNA footprinting experiments on the top and bottom strands of the T7 A2 promoter. Reactive thymines are indicated by arrows. DNase I-protected and KMnO 4 -sensitive sites found in the mutant and the wt promoter complexes are shown in black. DNase I-protected and KMnO 4 -sensitive sites specific for the wt complex are shown in gray. ( C ) Heparin resistance of RNAP-T7 A2 promoter complexes. Promoter complexes were formed as described above, and heparin was added to the final concentration 200 μg/ml (time 0), and incubation at 37°C was continued. At various time points, reaction aliquots were withdrawn, probed with KMnO 4 , resolved by denaturing PAGE, and quantified by using a PhosphorImager (Molecular Dynamics). The mean values of three independent measurements are given. The error bars represent the SD of the measurements. The normalized opening (%) value was calculated as described in Materials and Methods . ( D ) Reactions were set up as described above in a buffer containing the indicated concentrations of NaCl. Reactions were incubated for 15 min at 37°C and analyzed as in C .

    Techniques Used: Mutagenesis, Labeling, Sequencing, Autoradiography, DNA Footprinting, Concentration Assay, Incubation, Polyacrylamide Gel Electrophoresis

    4) Product Images from "A mutant RNA polymerase that forms unusual open promoter complexes"

    Article Title: A mutant RNA polymerase that forms unusual open promoter complexes

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    The mutant RNAP promoter complexes are stable at low temperatures. ( A ) DNase I footprinting and KMnO 4 probing of promoter complexes at different temperatures (bottom strand). Promoter complexes were formed at the indicated temperatures and followed by DNase I and KMnO 4 treatment. ( B ) KMnO 4 probing of promoter complexes formed at −20°C in 50% glycerol to prevent freezing. The 37°C controls were conducted in the presence (lanes 4 and 6) or the absence (lanes 3 and 5) of 50% glycerol in the reaction buffer. The results indicate that open complex formation and KMnO 4 sensitivity were not affected by the glycerol. See Materials and Methods for experimental details. ( C ) Quantitative KMnO 4 probing of promoter complexes formed at different temperatures. Promoter complex formation reactions were set up on ice or 37°C, brought to the assay temperature, incubated for 15 min, and probed with KMnO 4 . The reaction products were separated by denaturing PAGE ( Top ) and quantified as described in Materials and Methods ( Bottom ). KMnO 4 probing was performed at 0 (lanes 1, 12, 13, and 24); 5 (lanes 2, 11, 14, and 23); 10 (lanes 3, 10, 15, and 22); 15 (lanes 4, 9, 16, and 21); 23 (lanes 5, 8, 17, and 20); and 37°C (lanes 6, 7, 18, and 19). The upward vertical arrows indicate an up-temperature shift from 0°C to the assay temperature; downward arrows indicate a down-temperature shift from 37°C to the assay temperature.
    Figure Legend Snippet: The mutant RNAP promoter complexes are stable at low temperatures. ( A ) DNase I footprinting and KMnO 4 probing of promoter complexes at different temperatures (bottom strand). Promoter complexes were formed at the indicated temperatures and followed by DNase I and KMnO 4 treatment. ( B ) KMnO 4 probing of promoter complexes formed at −20°C in 50% glycerol to prevent freezing. The 37°C controls were conducted in the presence (lanes 4 and 6) or the absence (lanes 3 and 5) of 50% glycerol in the reaction buffer. The results indicate that open complex formation and KMnO 4 sensitivity were not affected by the glycerol. See Materials and Methods for experimental details. ( C ) Quantitative KMnO 4 probing of promoter complexes formed at different temperatures. Promoter complex formation reactions were set up on ice or 37°C, brought to the assay temperature, incubated for 15 min, and probed with KMnO 4 . The reaction products were separated by denaturing PAGE ( Top ) and quantified as described in Materials and Methods ( Bottom ). KMnO 4 probing was performed at 0 (lanes 1, 12, 13, and 24); 5 (lanes 2, 11, 14, and 23); 10 (lanes 3, 10, 15, and 22); 15 (lanes 4, 9, 16, and 21); 23 (lanes 5, 8, 17, and 20); and 37°C (lanes 6, 7, 18, and 19). The upward vertical arrows indicate an up-temperature shift from 0°C to the assay temperature; downward arrows indicate a down-temperature shift from 37°C to the assay temperature.

    Techniques Used: Mutagenesis, Footprinting, Incubation, Polyacrylamide Gel Electrophoresis

    The mutant RNAP forms stable promoter complexes that lack protein–DNA contacts downstream of the transcription initiation start point. ( A ) 100 nM of a 106-bp DNA fragment containing the T7 A2 promoter (−84 to +32) 32 P-end-labeled on the bottom strand was combined with 200 nM wt or mutant RNAP in 20-μl reactions containing 40 mM Tris⋅HCl (pH 7.9), 40 mM KCl, and 10 mM MgCl 2 . Reactions were preincubated for 15 min at 37°C and footprinted with DNase I or probed with KMnO 4 . Reaction products were resolved on a 6% sequencing gel and visualized by autoradiography. ( B ) Summary of the RNAP–DNA footprinting experiments on the top and bottom strands of the T7 A2 promoter. Reactive thymines are indicated by arrows. DNase I-protected and KMnO 4 -sensitive sites found in the mutant and the wt promoter complexes are shown in black. DNase I-protected and KMnO 4 -sensitive sites specific for the wt complex are shown in gray. ( C ) Heparin resistance of RNAP-T7 A2 promoter complexes. Promoter complexes were formed as described above, and heparin was added to the final concentration 200 μg/ml (time 0), and incubation at 37°C was continued. At various time points, reaction aliquots were withdrawn, probed with KMnO 4 , resolved by denaturing PAGE, and quantified by using a PhosphorImager (Molecular Dynamics). The mean values of three independent measurements are given. The error bars represent the SD of the measurements. The normalized opening (%) value was calculated as described in Materials and Methods . ( D ) Reactions were set up as described above in a buffer containing the indicated concentrations of NaCl. Reactions were incubated for 15 min at 37°C and analyzed as in C .
    Figure Legend Snippet: The mutant RNAP forms stable promoter complexes that lack protein–DNA contacts downstream of the transcription initiation start point. ( A ) 100 nM of a 106-bp DNA fragment containing the T7 A2 promoter (−84 to +32) 32 P-end-labeled on the bottom strand was combined with 200 nM wt or mutant RNAP in 20-μl reactions containing 40 mM Tris⋅HCl (pH 7.9), 40 mM KCl, and 10 mM MgCl 2 . Reactions were preincubated for 15 min at 37°C and footprinted with DNase I or probed with KMnO 4 . Reaction products were resolved on a 6% sequencing gel and visualized by autoradiography. ( B ) Summary of the RNAP–DNA footprinting experiments on the top and bottom strands of the T7 A2 promoter. Reactive thymines are indicated by arrows. DNase I-protected and KMnO 4 -sensitive sites found in the mutant and the wt promoter complexes are shown in black. DNase I-protected and KMnO 4 -sensitive sites specific for the wt complex are shown in gray. ( C ) Heparin resistance of RNAP-T7 A2 promoter complexes. Promoter complexes were formed as described above, and heparin was added to the final concentration 200 μg/ml (time 0), and incubation at 37°C was continued. At various time points, reaction aliquots were withdrawn, probed with KMnO 4 , resolved by denaturing PAGE, and quantified by using a PhosphorImager (Molecular Dynamics). The mean values of three independent measurements are given. The error bars represent the SD of the measurements. The normalized opening (%) value was calculated as described in Materials and Methods . ( D ) Reactions were set up as described above in a buffer containing the indicated concentrations of NaCl. Reactions were incubated for 15 min at 37°C and analyzed as in C .

    Techniques Used: Mutagenesis, Labeling, Sequencing, Autoradiography, DNA Footprinting, Concentration Assay, Incubation, Polyacrylamide Gel Electrophoresis

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