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New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex.

Doniselli N, Rodriguez-Aliaga P, Amidani D, Bardales JA, Bustamante C, Guerra DG, Rivetti C - Nucleic Acids Res. (2015)

Bottom Line: Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner.Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site.At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy.

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In vitro single-round transcription assays. (A) rrnB P1 promoter activity with 1X iNTPs (1 mM ATP, 0.1 mM CTP) and with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4); without iNTPs (lane 5). (B) rrnB P1 promoter activity with 10X iNTPs (10 mM ATP, 1 mM CTP) and no modulators (lane 1); with 10X iNTPs and 650 nM DksA (lane 2); with 0.1X iNTPs (0.1 mM ATP, 0.01 mM CTP) and no modulators (lane 3); with 0.1X iNTPs and 650 nM DksA (lane 4). (C) Plot of the rrnB P1 relative promoter activity (RPA) in the presence of 650 nM DksA versus the iNTPs concentration. (D) rrnB P1 (dis) promoter activity with 1X iNTPs and with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4). (E) λ pR promoter activity with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4); with 200 μM ppGpp added after RPo formation (lane 5). All transcription reactions were carried out in the presence 100 μg/ml heparin. The two bands of the transcript are probably due to inhomogeneous run-off termination. For each gel, the RPA indicated below each lane was determined from the cumulative intensity of the two bands relative to that in lane 1 except for lane 4 in panel B which is relative to lane 3.
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Figure 8: In vitro single-round transcription assays. (A) rrnB P1 promoter activity with 1X iNTPs (1 mM ATP, 0.1 mM CTP) and with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4); without iNTPs (lane 5). (B) rrnB P1 promoter activity with 10X iNTPs (10 mM ATP, 1 mM CTP) and no modulators (lane 1); with 10X iNTPs and 650 nM DksA (lane 2); with 0.1X iNTPs (0.1 mM ATP, 0.01 mM CTP) and no modulators (lane 3); with 0.1X iNTPs and 650 nM DksA (lane 4). (C) Plot of the rrnB P1 relative promoter activity (RPA) in the presence of 650 nM DksA versus the iNTPs concentration. (D) rrnB P1 (dis) promoter activity with 1X iNTPs and with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4). (E) λ pR promoter activity with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4); with 200 μM ppGpp added after RPo formation (lane 5). All transcription reactions were carried out in the presence 100 μg/ml heparin. The two bands of the transcript are probably due to inhomogeneous run-off termination. For each gel, the RPA indicated below each lane was determined from the cumulative intensity of the two bands relative to that in lane 1 except for lane 4 in panel B which is relative to lane 3.

Mentions: Transcription reactions were assembled in 10 μl final volume as follows: RNAP (35 nM) was preincubated in transcription buffer with none, one or both modulators at the concentrations indicated in Figure 8, for 10 min at 37°C. Afterward, DNA templates (20 nM) and iNTPs (as indicated in Figure 8) were added and the reaction was incubated for 15 min at 37°C to favor RPo formation. Transcription was started by the addition of 1 μl NTP mix composed of 200 μM ATP, CTP and GTP, 100 μM UTP and 1 mg/ml heparin immediately after the addition of [α-32P]UTP (1 μCi; 1 Ci = 37 GBq). After 10 min of incubation at 30°C the reaction was terminated by the addition of 10 μl of formamide loading buffer. Electrophoresis was performed on a 7 M urea-6% polyacrylamide gel and the RNA transcript was quantified by a Packard Cyclone (Alliance Analytical). The in vitro transcription assays were repeated at least two times.


New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex.

Doniselli N, Rodriguez-Aliaga P, Amidani D, Bardales JA, Bustamante C, Guerra DG, Rivetti C - Nucleic Acids Res. (2015)

In vitro single-round transcription assays. (A) rrnB P1 promoter activity with 1X iNTPs (1 mM ATP, 0.1 mM CTP) and with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4); without iNTPs (lane 5). (B) rrnB P1 promoter activity with 10X iNTPs (10 mM ATP, 1 mM CTP) and no modulators (lane 1); with 10X iNTPs and 650 nM DksA (lane 2); with 0.1X iNTPs (0.1 mM ATP, 0.01 mM CTP) and no modulators (lane 3); with 0.1X iNTPs and 650 nM DksA (lane 4). (C) Plot of the rrnB P1 relative promoter activity (RPA) in the presence of 650 nM DksA versus the iNTPs concentration. (D) rrnB P1 (dis) promoter activity with 1X iNTPs and with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4). (E) λ pR promoter activity with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4); with 200 μM ppGpp added after RPo formation (lane 5). All transcription reactions were carried out in the presence 100 μg/ml heparin. The two bands of the transcript are probably due to inhomogeneous run-off termination. For each gel, the RPA indicated below each lane was determined from the cumulative intensity of the two bands relative to that in lane 1 except for lane 4 in panel B which is relative to lane 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 8: In vitro single-round transcription assays. (A) rrnB P1 promoter activity with 1X iNTPs (1 mM ATP, 0.1 mM CTP) and with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4); without iNTPs (lane 5). (B) rrnB P1 promoter activity with 10X iNTPs (10 mM ATP, 1 mM CTP) and no modulators (lane 1); with 10X iNTPs and 650 nM DksA (lane 2); with 0.1X iNTPs (0.1 mM ATP, 0.01 mM CTP) and no modulators (lane 3); with 0.1X iNTPs and 650 nM DksA (lane 4). (C) Plot of the rrnB P1 relative promoter activity (RPA) in the presence of 650 nM DksA versus the iNTPs concentration. (D) rrnB P1 (dis) promoter activity with 1X iNTPs and with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4). (E) λ pR promoter activity with no modulators (lane 1); with 200 μM ppGpp (lane 2); with 650 nM DksA (lane 3); with 200 μM ppGpp and 650 nM DksA (lane 4); with 200 μM ppGpp added after RPo formation (lane 5). All transcription reactions were carried out in the presence 100 μg/ml heparin. The two bands of the transcript are probably due to inhomogeneous run-off termination. For each gel, the RPA indicated below each lane was determined from the cumulative intensity of the two bands relative to that in lane 1 except for lane 4 in panel B which is relative to lane 3.
Mentions: Transcription reactions were assembled in 10 μl final volume as follows: RNAP (35 nM) was preincubated in transcription buffer with none, one or both modulators at the concentrations indicated in Figure 8, for 10 min at 37°C. Afterward, DNA templates (20 nM) and iNTPs (as indicated in Figure 8) were added and the reaction was incubated for 15 min at 37°C to favor RPo formation. Transcription was started by the addition of 1 μl NTP mix composed of 200 μM ATP, CTP and GTP, 100 μM UTP and 1 mg/ml heparin immediately after the addition of [α-32P]UTP (1 μCi; 1 Ci = 37 GBq). After 10 min of incubation at 30°C the reaction was terminated by the addition of 10 μl of formamide loading buffer. Electrophoresis was performed on a 7 M urea-6% polyacrylamide gel and the RNA transcript was quantified by a Packard Cyclone (Alliance Analytical). The in vitro transcription assays were repeated at least two times.

Bottom Line: Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner.Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site.At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy.

Show MeSH
Related in: MedlinePlus