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    1) Product Images from "Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli"

    Article Title: Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

    Journal: bioRxiv

    doi: 10.1101/2021.04.06.438613

    Distributions of 5′-end sequences for RNAs generated in primer-independent initiation and primer-dependent initiation  in vivo  and  in vitro . A. lac CONS-N10 library. Base pairs in the N10 region are numbered based on their position relative to the promoter −10 element. Colors as in   Figure 1B . B-C.  RNA 5′-end distribution histograms (mean ± SD, N = 3) for RNAs generated by primer-independent initiation (top) or primer-dependent initiation (bottom) in stationary-phase  E. coli  cells (panel B) or  in vitro , with the dinucleotide primer UpA (panel C). Dashed line indicates the mean 5′-end position (mean ± SD, N = 3).
    Figure Legend Snippet: Distributions of 5′-end sequences for RNAs generated in primer-independent initiation and primer-dependent initiation in vivo and in vitro . A. lac CONS-N10 library. Base pairs in the N10 region are numbered based on their position relative to the promoter −10 element. Colors as in Figure 1B . B-C. RNA 5′-end distribution histograms (mean ± SD, N = 3) for RNAs generated by primer-independent initiation (top) or primer-dependent initiation (bottom) in stationary-phase E. coli cells (panel B) or in vitro , with the dinucleotide primer UpA (panel C). Dashed line indicates the mean 5′-end position (mean ± SD, N = 3).

    Techniques Used: Generated, In Vivo, In Vitro

    Promoter-sequence dependence of primer-dependent initiation  in vivo : sequences flanking the primer binding site Sequence logo (  35 ) for primer-dependent initiation in stationary-phase  E. coli  cells with each of the 16 dinucleotides at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively). The height of each base “X” at each position “Y” represents the log 2  average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Gray box indicates positions where enrichment values could not be computed.
    Figure Legend Snippet: Promoter-sequence dependence of primer-dependent initiation in vivo : sequences flanking the primer binding site Sequence logo ( 35 ) for primer-dependent initiation in stationary-phase E. coli cells with each of the 16 dinucleotides at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively). The height of each base “X” at each position “Y” represents the log 2 average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Gray box indicates positions where enrichment values could not be computed.

    Techniques Used: Sequencing, In Vivo, Binding Assay

    Promoter-sequence dependence of primer-dependent initiation: primer binding site. A.  Relative usage of dinucleotides in primer-dependent initiation in stationary-phase  E. coli  cells. Values represent the percentage of total 5′-OH RNAs generated using each of the 16 dinucleotide primers (mean, N = 3). Bold, dinucleotides preferentially used as primers. B.  Complementarity between the primer binding site and dinucleotide in primer-dependent initiation. Top: primer-dependent initiation involving template-strand complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS), template-strand complementarity to only 3′ nucleotide of primer (TSS), template-strand complementarity to only 5′ nucleotide of primer (TSS-1), or no template-strand complementarity to primer (none). Three vertical lines, complementarity; X, non-complementarity. Other symbols and colors as in   Figure 1 . Bottom: percentage of primer-dependent initiation involving complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS; pink), complementarity to only 3′ nucleotide of primer (TSS; purple), or template-strand complementarity to only 5′ nucleotide of primer or no template-strand complementarity to primer (TSS-1 or none; white) in stationary-phase  E. coli  cells (left) or  in vitro , with the dinucleotide primer UpA (right) (mean ± SD, N = 3).
    Figure Legend Snippet: Promoter-sequence dependence of primer-dependent initiation: primer binding site. A. Relative usage of dinucleotides in primer-dependent initiation in stationary-phase E. coli cells. Values represent the percentage of total 5′-OH RNAs generated using each of the 16 dinucleotide primers (mean, N = 3). Bold, dinucleotides preferentially used as primers. B. Complementarity between the primer binding site and dinucleotide in primer-dependent initiation. Top: primer-dependent initiation involving template-strand complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS), template-strand complementarity to only 3′ nucleotide of primer (TSS), template-strand complementarity to only 5′ nucleotide of primer (TSS-1), or no template-strand complementarity to primer (none). Three vertical lines, complementarity; X, non-complementarity. Other symbols and colors as in Figure 1 . Bottom: percentage of primer-dependent initiation involving complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS; pink), complementarity to only 3′ nucleotide of primer (TSS; purple), or template-strand complementarity to only 5′ nucleotide of primer or no template-strand complementarity to primer (TSS-1 or none; white) in stationary-phase E. coli cells (left) or in vitro , with the dinucleotide primer UpA (right) (mean ± SD, N = 3).

    Techniques Used: Sequencing, Binding Assay, Generated, In Vitro

    Promoter-sequence dependence of primer-dependent initiation in stationary-phase  E. coli  cells: primer binding site Top: primer-dependent initiation involving template-strand complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS), template-strand complementarity to only 3′ nucleotide of primer (TSS), template-strand complementarity to only 5′ nucleotide of primer (TSS-1), or no template-strand complementarity to primer (none). Three vertical lines, complementarity; X, non-complementarity. Other symbols and colors as in   Figure 1 . Bottom: percentage of primer-dependent initiation involving complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS; pink), complementarity to only 3′ nucleotide of primer (TSS; purple), or template-strand complementarity to only 5′ nucleotide of primer or no template-strand complementarity to primer (TSS-1 or none; white) in stationary-phase  E. coli  cells for primer binding sites located 6-7, 7-8, or 8-9 base pairs downstream of the promoter −10 element (mean ± SD, N = 3).
    Figure Legend Snippet: Promoter-sequence dependence of primer-dependent initiation in stationary-phase E. coli cells: primer binding site Top: primer-dependent initiation involving template-strand complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS), template-strand complementarity to only 3′ nucleotide of primer (TSS), template-strand complementarity to only 5′ nucleotide of primer (TSS-1), or no template-strand complementarity to primer (none). Three vertical lines, complementarity; X, non-complementarity. Other symbols and colors as in Figure 1 . Bottom: percentage of primer-dependent initiation involving complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS; pink), complementarity to only 3′ nucleotide of primer (TSS; purple), or template-strand complementarity to only 5′ nucleotide of primer or no template-strand complementarity to primer (TSS-1 or none; white) in stationary-phase E. coli cells for primer binding sites located 6-7, 7-8, or 8-9 base pairs downstream of the promoter −10 element (mean ± SD, N = 3).

    Techniques Used: Sequencing, Binding Assay

    Promoter-sequence dependence of primer-dependent initiation: sequences flanking the primer binding site. Sequence logo (  35 ) for primer-dependent initiation at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively) in stationary-phase  E. coli  cells (left) or  in vitro , with the dinucleotide primer UpA (right). The height of each base “X” at each position “Y” represents the log 2  average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Other symbols and colors as in   Figure 1 .
    Figure Legend Snippet: Promoter-sequence dependence of primer-dependent initiation: sequences flanking the primer binding site. Sequence logo ( 35 ) for primer-dependent initiation at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively) in stationary-phase E. coli cells (left) or in vitro , with the dinucleotide primer UpA (right). The height of each base “X” at each position “Y” represents the log 2 average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Other symbols and colors as in Figure 1 .

    Techniques Used: Sequencing, Binding Assay, In Vitro

    Promoter-sequence dependence of primer-dependent initiation: chromosomal promoters A.  Sequence logo (  35 ) for primer-dependent initiation at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively) in stationary-phase  E. coli  cells for 93 natural, chromosomally-encoded promoters that use UpA as a primer. The height of each base “X” at each position “Y” represents the log 2  average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Other symbols and colors as in   Figure 1 . B.  Promoter-sequence dependence of primer-dependent initiation at the  E. coli bhsA  promoter. Top: sequences of DNA templates containing wild-type and mutant derivatives of  bhsA  promoter. Bottom: primer extension analysis of 5′-end lengths of  bhsA  RNAs. In primer-dependent initiation with a dinucleotide primer, the RNA product acquires one additional nucleotide at the RNA 5′ end (  Figure 1 ). Gel shows radiolabeled cDNA products derived from primer-independent initiation (5′-ppp) and primer-dependent initiation (5′-OH) in stationary-phase  E. coli  cells. Bottom right: ratios of primer-dependent initiation vs. primer-independent initiation (mean ± SD, N = 4).
    Figure Legend Snippet: Promoter-sequence dependence of primer-dependent initiation: chromosomal promoters A. Sequence logo ( 35 ) for primer-dependent initiation at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively) in stationary-phase E. coli cells for 93 natural, chromosomally-encoded promoters that use UpA as a primer. The height of each base “X” at each position “Y” represents the log 2 average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Other symbols and colors as in Figure 1 . B. Promoter-sequence dependence of primer-dependent initiation at the E. coli bhsA promoter. Top: sequences of DNA templates containing wild-type and mutant derivatives of bhsA promoter. Bottom: primer extension analysis of 5′-end lengths of bhsA RNAs. In primer-dependent initiation with a dinucleotide primer, the RNA product acquires one additional nucleotide at the RNA 5′ end ( Figure 1 ). Gel shows radiolabeled cDNA products derived from primer-independent initiation (5′-ppp) and primer-dependent initiation (5′-OH) in stationary-phase E. coli cells. Bottom right: ratios of primer-dependent initiation vs. primer-independent initiation (mean ± SD, N = 4).

    Techniques Used: Sequencing, Binding Assay, Mutagenesis, Derivative Assay

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    Electrophoresis:

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    Isolation:

    Article Title: Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli
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    Incubation:

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    In Vitro:

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    Concentration Assay:

    Article Title: Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli
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    Ligation:

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    Purification:

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    Transformation Assay:

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    New England Biolabs stationary phase e coli cells
    Distributions of 5′-end sequences for RNAs generated in primer-independent initiation and primer-dependent initiation  in vivo  and  in vitro . A. lac CONS-N10 library. Base pairs in the N10 region are numbered based on their position relative to the promoter −10 element. Colors as in   Figure 1B . B-C.  RNA 5′-end distribution histograms (mean ± SD, N = 3) for RNAs generated by primer-independent initiation (top) or primer-dependent initiation (bottom) in stationary-phase  E. coli  cells (panel B) or  in vitro , with the dinucleotide primer UpA (panel C). Dashed line indicates the mean 5′-end position (mean ± SD, N = 3).
    Stationary Phase E Coli Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/stationary phase e coli cells/product/New England Biolabs
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    stationary phase e coli cells - by Bioz Stars, 2021-05
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    Distributions of 5′-end sequences for RNAs generated in primer-independent initiation and primer-dependent initiation  in vivo  and  in vitro . A. lac CONS-N10 library. Base pairs in the N10 region are numbered based on their position relative to the promoter −10 element. Colors as in   Figure 1B . B-C.  RNA 5′-end distribution histograms (mean ± SD, N = 3) for RNAs generated by primer-independent initiation (top) or primer-dependent initiation (bottom) in stationary-phase  E. coli  cells (panel B) or  in vitro , with the dinucleotide primer UpA (panel C). Dashed line indicates the mean 5′-end position (mean ± SD, N = 3).

    Journal: bioRxiv

    Article Title: Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

    doi: 10.1101/2021.04.06.438613

    Figure Lengend Snippet: Distributions of 5′-end sequences for RNAs generated in primer-independent initiation and primer-dependent initiation in vivo and in vitro . A. lac CONS-N10 library. Base pairs in the N10 region are numbered based on their position relative to the promoter −10 element. Colors as in Figure 1B . B-C. RNA 5′-end distribution histograms (mean ± SD, N = 3) for RNAs generated by primer-independent initiation (top) or primer-dependent initiation (bottom) in stationary-phase E. coli cells (panel B) or in vitro , with the dinucleotide primer UpA (panel C). Dashed line indicates the mean 5′-end position (mean ± SD, N = 3).

    Article Snippet: Processed RNA products isolated from stationary-phase E. coli cells (in 10.5 μl of nuclease-free water) were combined with 1 mM ATP (NEB), 40 U RNaseOUT, 1x T4 RNA ligase buffer (NEB), and 10 U T4 RNA ligase 1 (NEB) and 1 μM of 5’ adaptor oligo (total reaction volume = 20 μl), and incubated at 37°C for 2 h. Reactions were then supplemented with 1x T4 RNA ligase buffer, 1 mM ATP, PEG 8000 (10% final), 5U T4 RNA ligase 1, and 20 U RNaseOUT (total reaction volume = 30 μl) and further incubated at 16°C for 16 h. Processed RNA products isolated from in vitro reactions (in 10.5 μl of nuclease-free water) were combined with PEG 8000 (10% final concentration), 1 mM ATP, 40 U RNaseOUT, 1x T4 RNA ligase buffer, 10 U T4 RNA ligase 1, and 1 μM of 5’ adaptor oligo (total reaction volume = 30 μl), and incubated at 16°C for 16 h. Ligation reactions were stopped by addition of 30 μl of 2x RNA loading dye and heated at 95°C for 5 min. For each replicate, the 4 ligation reactions were combined, and separated by electrophoresis on 10% 7M urea slab gels (equilibrated and run in 1x TBE).

    Techniques: Generated, In Vivo, In Vitro

    Promoter-sequence dependence of primer-dependent initiation  in vivo : sequences flanking the primer binding site Sequence logo (  35 ) for primer-dependent initiation in stationary-phase  E. coli  cells with each of the 16 dinucleotides at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively). The height of each base “X” at each position “Y” represents the log 2  average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Gray box indicates positions where enrichment values could not be computed.

    Journal: bioRxiv

    Article Title: Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

    doi: 10.1101/2021.04.06.438613

    Figure Lengend Snippet: Promoter-sequence dependence of primer-dependent initiation in vivo : sequences flanking the primer binding site Sequence logo ( 35 ) for primer-dependent initiation in stationary-phase E. coli cells with each of the 16 dinucleotides at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively). The height of each base “X” at each position “Y” represents the log 2 average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Gray box indicates positions where enrichment values could not be computed.

    Article Snippet: Processed RNA products isolated from stationary-phase E. coli cells (in 10.5 μl of nuclease-free water) were combined with 1 mM ATP (NEB), 40 U RNaseOUT, 1x T4 RNA ligase buffer (NEB), and 10 U T4 RNA ligase 1 (NEB) and 1 μM of 5’ adaptor oligo (total reaction volume = 20 μl), and incubated at 37°C for 2 h. Reactions were then supplemented with 1x T4 RNA ligase buffer, 1 mM ATP, PEG 8000 (10% final), 5U T4 RNA ligase 1, and 20 U RNaseOUT (total reaction volume = 30 μl) and further incubated at 16°C for 16 h. Processed RNA products isolated from in vitro reactions (in 10.5 μl of nuclease-free water) were combined with PEG 8000 (10% final concentration), 1 mM ATP, 40 U RNaseOUT, 1x T4 RNA ligase buffer, 10 U T4 RNA ligase 1, and 1 μM of 5’ adaptor oligo (total reaction volume = 30 μl), and incubated at 16°C for 16 h. Ligation reactions were stopped by addition of 30 μl of 2x RNA loading dye and heated at 95°C for 5 min. For each replicate, the 4 ligation reactions were combined, and separated by electrophoresis on 10% 7M urea slab gels (equilibrated and run in 1x TBE).

    Techniques: Sequencing, In Vivo, Binding Assay

    Promoter-sequence dependence of primer-dependent initiation: primer binding site. A.  Relative usage of dinucleotides in primer-dependent initiation in stationary-phase  E. coli  cells. Values represent the percentage of total 5′-OH RNAs generated using each of the 16 dinucleotide primers (mean, N = 3). Bold, dinucleotides preferentially used as primers. B.  Complementarity between the primer binding site and dinucleotide in primer-dependent initiation. Top: primer-dependent initiation involving template-strand complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS), template-strand complementarity to only 3′ nucleotide of primer (TSS), template-strand complementarity to only 5′ nucleotide of primer (TSS-1), or no template-strand complementarity to primer (none). Three vertical lines, complementarity; X, non-complementarity. Other symbols and colors as in   Figure 1 . Bottom: percentage of primer-dependent initiation involving complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS; pink), complementarity to only 3′ nucleotide of primer (TSS; purple), or template-strand complementarity to only 5′ nucleotide of primer or no template-strand complementarity to primer (TSS-1 or none; white) in stationary-phase  E. coli  cells (left) or  in vitro , with the dinucleotide primer UpA (right) (mean ± SD, N = 3).

    Journal: bioRxiv

    Article Title: Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

    doi: 10.1101/2021.04.06.438613

    Figure Lengend Snippet: Promoter-sequence dependence of primer-dependent initiation: primer binding site. A. Relative usage of dinucleotides in primer-dependent initiation in stationary-phase E. coli cells. Values represent the percentage of total 5′-OH RNAs generated using each of the 16 dinucleotide primers (mean, N = 3). Bold, dinucleotides preferentially used as primers. B. Complementarity between the primer binding site and dinucleotide in primer-dependent initiation. Top: primer-dependent initiation involving template-strand complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS), template-strand complementarity to only 3′ nucleotide of primer (TSS), template-strand complementarity to only 5′ nucleotide of primer (TSS-1), or no template-strand complementarity to primer (none). Three vertical lines, complementarity; X, non-complementarity. Other symbols and colors as in Figure 1 . Bottom: percentage of primer-dependent initiation involving complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS; pink), complementarity to only 3′ nucleotide of primer (TSS; purple), or template-strand complementarity to only 5′ nucleotide of primer or no template-strand complementarity to primer (TSS-1 or none; white) in stationary-phase E. coli cells (left) or in vitro , with the dinucleotide primer UpA (right) (mean ± SD, N = 3).

    Article Snippet: Processed RNA products isolated from stationary-phase E. coli cells (in 10.5 μl of nuclease-free water) were combined with 1 mM ATP (NEB), 40 U RNaseOUT, 1x T4 RNA ligase buffer (NEB), and 10 U T4 RNA ligase 1 (NEB) and 1 μM of 5’ adaptor oligo (total reaction volume = 20 μl), and incubated at 37°C for 2 h. Reactions were then supplemented with 1x T4 RNA ligase buffer, 1 mM ATP, PEG 8000 (10% final), 5U T4 RNA ligase 1, and 20 U RNaseOUT (total reaction volume = 30 μl) and further incubated at 16°C for 16 h. Processed RNA products isolated from in vitro reactions (in 10.5 μl of nuclease-free water) were combined with PEG 8000 (10% final concentration), 1 mM ATP, 40 U RNaseOUT, 1x T4 RNA ligase buffer, 10 U T4 RNA ligase 1, and 1 μM of 5’ adaptor oligo (total reaction volume = 30 μl), and incubated at 16°C for 16 h. Ligation reactions were stopped by addition of 30 μl of 2x RNA loading dye and heated at 95°C for 5 min. For each replicate, the 4 ligation reactions were combined, and separated by electrophoresis on 10% 7M urea slab gels (equilibrated and run in 1x TBE).

    Techniques: Sequencing, Binding Assay, Generated, In Vitro

    Promoter-sequence dependence of primer-dependent initiation in stationary-phase  E. coli  cells: primer binding site Top: primer-dependent initiation involving template-strand complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS), template-strand complementarity to only 3′ nucleotide of primer (TSS), template-strand complementarity to only 5′ nucleotide of primer (TSS-1), or no template-strand complementarity to primer (none). Three vertical lines, complementarity; X, non-complementarity. Other symbols and colors as in   Figure 1 . Bottom: percentage of primer-dependent initiation involving complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS; pink), complementarity to only 3′ nucleotide of primer (TSS; purple), or template-strand complementarity to only 5′ nucleotide of primer or no template-strand complementarity to primer (TSS-1 or none; white) in stationary-phase  E. coli  cells for primer binding sites located 6-7, 7-8, or 8-9 base pairs downstream of the promoter −10 element (mean ± SD, N = 3).

    Journal: bioRxiv

    Article Title: Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

    doi: 10.1101/2021.04.06.438613

    Figure Lengend Snippet: Promoter-sequence dependence of primer-dependent initiation in stationary-phase E. coli cells: primer binding site Top: primer-dependent initiation involving template-strand complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS), template-strand complementarity to only 3′ nucleotide of primer (TSS), template-strand complementarity to only 5′ nucleotide of primer (TSS-1), or no template-strand complementarity to primer (none). Three vertical lines, complementarity; X, non-complementarity. Other symbols and colors as in Figure 1 . Bottom: percentage of primer-dependent initiation involving complementarity to both 5′ and 3′ nucleotides of primer (TSS-1, TSS; pink), complementarity to only 3′ nucleotide of primer (TSS; purple), or template-strand complementarity to only 5′ nucleotide of primer or no template-strand complementarity to primer (TSS-1 or none; white) in stationary-phase E. coli cells for primer binding sites located 6-7, 7-8, or 8-9 base pairs downstream of the promoter −10 element (mean ± SD, N = 3).

    Article Snippet: Processed RNA products isolated from stationary-phase E. coli cells (in 10.5 μl of nuclease-free water) were combined with 1 mM ATP (NEB), 40 U RNaseOUT, 1x T4 RNA ligase buffer (NEB), and 10 U T4 RNA ligase 1 (NEB) and 1 μM of 5’ adaptor oligo (total reaction volume = 20 μl), and incubated at 37°C for 2 h. Reactions were then supplemented with 1x T4 RNA ligase buffer, 1 mM ATP, PEG 8000 (10% final), 5U T4 RNA ligase 1, and 20 U RNaseOUT (total reaction volume = 30 μl) and further incubated at 16°C for 16 h. Processed RNA products isolated from in vitro reactions (in 10.5 μl of nuclease-free water) were combined with PEG 8000 (10% final concentration), 1 mM ATP, 40 U RNaseOUT, 1x T4 RNA ligase buffer, 10 U T4 RNA ligase 1, and 1 μM of 5’ adaptor oligo (total reaction volume = 30 μl), and incubated at 16°C for 16 h. Ligation reactions were stopped by addition of 30 μl of 2x RNA loading dye and heated at 95°C for 5 min. For each replicate, the 4 ligation reactions were combined, and separated by electrophoresis on 10% 7M urea slab gels (equilibrated and run in 1x TBE).

    Techniques: Sequencing, Binding Assay

    Promoter-sequence dependence of primer-dependent initiation: sequences flanking the primer binding site. Sequence logo (  35 ) for primer-dependent initiation at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively) in stationary-phase  E. coli  cells (left) or  in vitro , with the dinucleotide primer UpA (right). The height of each base “X” at each position “Y” represents the log 2  average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Other symbols and colors as in   Figure 1 .

    Journal: bioRxiv

    Article Title: Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

    doi: 10.1101/2021.04.06.438613

    Figure Lengend Snippet: Promoter-sequence dependence of primer-dependent initiation: sequences flanking the primer binding site. Sequence logo ( 35 ) for primer-dependent initiation at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively) in stationary-phase E. coli cells (left) or in vitro , with the dinucleotide primer UpA (right). The height of each base “X” at each position “Y” represents the log 2 average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Other symbols and colors as in Figure 1 .

    Article Snippet: Processed RNA products isolated from stationary-phase E. coli cells (in 10.5 μl of nuclease-free water) were combined with 1 mM ATP (NEB), 40 U RNaseOUT, 1x T4 RNA ligase buffer (NEB), and 10 U T4 RNA ligase 1 (NEB) and 1 μM of 5’ adaptor oligo (total reaction volume = 20 μl), and incubated at 37°C for 2 h. Reactions were then supplemented with 1x T4 RNA ligase buffer, 1 mM ATP, PEG 8000 (10% final), 5U T4 RNA ligase 1, and 20 U RNaseOUT (total reaction volume = 30 μl) and further incubated at 16°C for 16 h. Processed RNA products isolated from in vitro reactions (in 10.5 μl of nuclease-free water) were combined with PEG 8000 (10% final concentration), 1 mM ATP, 40 U RNaseOUT, 1x T4 RNA ligase buffer, 10 U T4 RNA ligase 1, and 1 μM of 5’ adaptor oligo (total reaction volume = 30 μl), and incubated at 16°C for 16 h. Ligation reactions were stopped by addition of 30 μl of 2x RNA loading dye and heated at 95°C for 5 min. For each replicate, the 4 ligation reactions were combined, and separated by electrophoresis on 10% 7M urea slab gels (equilibrated and run in 1x TBE).

    Techniques: Sequencing, Binding Assay, In Vitro

    Promoter-sequence dependence of primer-dependent initiation: chromosomal promoters A.  Sequence logo (  35 ) for primer-dependent initiation at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively) in stationary-phase  E. coli  cells for 93 natural, chromosomally-encoded promoters that use UpA as a primer. The height of each base “X” at each position “Y” represents the log 2  average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Other symbols and colors as in   Figure 1 . B.  Promoter-sequence dependence of primer-dependent initiation at the  E. coli bhsA  promoter. Top: sequences of DNA templates containing wild-type and mutant derivatives of  bhsA  promoter. Bottom: primer extension analysis of 5′-end lengths of  bhsA  RNAs. In primer-dependent initiation with a dinucleotide primer, the RNA product acquires one additional nucleotide at the RNA 5′ end (  Figure 1 ). Gel shows radiolabeled cDNA products derived from primer-independent initiation (5′-ppp) and primer-dependent initiation (5′-OH) in stationary-phase  E. coli  cells. Bottom right: ratios of primer-dependent initiation vs. primer-independent initiation (mean ± SD, N = 4).

    Journal: bioRxiv

    Article Title: Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

    doi: 10.1101/2021.04.06.438613

    Figure Lengend Snippet: Promoter-sequence dependence of primer-dependent initiation: chromosomal promoters A. Sequence logo ( 35 ) for primer-dependent initiation at TSS positions 7, 8, and 9 (corresponding to primer binding sites 6-7, 7-8, and 8-9, respectively) in stationary-phase E. coli cells for 93 natural, chromosomally-encoded promoters that use UpA as a primer. The height of each base “X” at each position “Y” represents the log 2 average of the % 5′-OH RNAs computed across sequences containing nontemplate-strand X at position Y. Red, consensus nucleotides; black, non-consensus nucleotides. Other symbols and colors as in Figure 1 . B. Promoter-sequence dependence of primer-dependent initiation at the E. coli bhsA promoter. Top: sequences of DNA templates containing wild-type and mutant derivatives of bhsA promoter. Bottom: primer extension analysis of 5′-end lengths of bhsA RNAs. In primer-dependent initiation with a dinucleotide primer, the RNA product acquires one additional nucleotide at the RNA 5′ end ( Figure 1 ). Gel shows radiolabeled cDNA products derived from primer-independent initiation (5′-ppp) and primer-dependent initiation (5′-OH) in stationary-phase E. coli cells. Bottom right: ratios of primer-dependent initiation vs. primer-independent initiation (mean ± SD, N = 4).

    Article Snippet: Processed RNA products isolated from stationary-phase E. coli cells (in 10.5 μl of nuclease-free water) were combined with 1 mM ATP (NEB), 40 U RNaseOUT, 1x T4 RNA ligase buffer (NEB), and 10 U T4 RNA ligase 1 (NEB) and 1 μM of 5’ adaptor oligo (total reaction volume = 20 μl), and incubated at 37°C for 2 h. Reactions were then supplemented with 1x T4 RNA ligase buffer, 1 mM ATP, PEG 8000 (10% final), 5U T4 RNA ligase 1, and 20 U RNaseOUT (total reaction volume = 30 μl) and further incubated at 16°C for 16 h. Processed RNA products isolated from in vitro reactions (in 10.5 μl of nuclease-free water) were combined with PEG 8000 (10% final concentration), 1 mM ATP, 40 U RNaseOUT, 1x T4 RNA ligase buffer, 10 U T4 RNA ligase 1, and 1 μM of 5’ adaptor oligo (total reaction volume = 30 μl), and incubated at 16°C for 16 h. Ligation reactions were stopped by addition of 30 μl of 2x RNA loading dye and heated at 95°C for 5 min. For each replicate, the 4 ligation reactions were combined, and separated by electrophoresis on 10% 7M urea slab gels (equilibrated and run in 1x TBE).

    Techniques: Sequencing, Binding Assay, Mutagenesis, Derivative Assay