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An aromatic residue switch in enhancer-dependent bacterial RNA polymerase controls transcription intermediate complex activity.

Wiesler SC, Weinzierl RO, Buck M - Nucleic Acids Res. (2013)

Bottom Line: A single tryptophan is ideally positioned to directly engage with the flipped out base of the non-template strand at the +1 site.Evidence suggests that this tryptophan (i) is involved in either forward translocation or DNA scrunching and (ii) in σ(54)-regulated promoters limits the transcription activity of at least one intermediate complex (RPi) before the formation of a fully functional RPo.Limiting RPi activity may be important in preventing the premature synthesis of abortive transcripts, suggesting its involvement in a general mechanism driving the RPi to RPo transition for transcription initiation.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK. s.wiesler@imperial.ac.uk

ABSTRACT
The formation of the open promoter complex (RPo) in which the melted DNA containing the transcription start site is located at the RNA polymerase (RNAP) catalytic centre is an obligatory step in the transcription of DNA into RNA catalyzed by RNAP. In the RPo, an extensive network of interactions is established between DNA, RNAP and the σ-factor and the formation of functional RPo occurs via a series of transcriptional intermediates (collectively 'RPi'). A single tryptophan is ideally positioned to directly engage with the flipped out base of the non-template strand at the +1 site. Evidence suggests that this tryptophan (i) is involved in either forward translocation or DNA scrunching and (ii) in σ(54)-regulated promoters limits the transcription activity of at least one intermediate complex (RPi) before the formation of a fully functional RPo. Limiting RPi activity may be important in preventing the premature synthesis of abortive transcripts, suggesting its involvement in a general mechanism driving the RPi to RPo transition for transcription initiation.

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Catalytic and binding properties (A) βW183 mutants are defective in nucleic acid binding and holoenzyme formation in comparison with wild-type RNAP (wt). The catalytic defects of the variants can in part be explained by the defects in scaffold binding and holoenzyme formation. The assays were performed as titrations at three different RNAP concentrations (25 nM, 50 nM, 100 nM). The activity for each mutant was calculated relative to the WT activity. The data represent average activities, and the error bars represent standard deviations. For each reaction, 33 nM of α32P-GTP was used. The GTP turnover into RNA achieved by the WT in a minimal scaffold assay (7 nt RNA) was 6 nM (25 nM RNAP), 9 nM (50 nM RNAP) or 17 nM (100 nM RNAP), respectively. (B) Schematic of the minimal scaffold assay measuring RNA elongation (minimal scaffold assay) and pyrophosphorolysis (PPi assay) activities. For minimal scaffold binding assays, the minimal scaffold was 32P-labelled, incubated with RNAP and complexes were separated on a native gel.
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gkt271-F2: Catalytic and binding properties (A) βW183 mutants are defective in nucleic acid binding and holoenzyme formation in comparison with wild-type RNAP (wt). The catalytic defects of the variants can in part be explained by the defects in scaffold binding and holoenzyme formation. The assays were performed as titrations at three different RNAP concentrations (25 nM, 50 nM, 100 nM). The activity for each mutant was calculated relative to the WT activity. The data represent average activities, and the error bars represent standard deviations. For each reaction, 33 nM of α32P-GTP was used. The GTP turnover into RNA achieved by the WT in a minimal scaffold assay (7 nt RNA) was 6 nM (25 nM RNAP), 9 nM (50 nM RNAP) or 17 nM (100 nM RNAP), respectively. (B) Schematic of the minimal scaffold assay measuring RNA elongation (minimal scaffold assay) and pyrophosphorolysis (PPi assay) activities. For minimal scaffold binding assays, the minimal scaffold was 32P-labelled, incubated with RNAP and complexes were separated on a native gel.

Mentions: Single residue substitutions were created at position βW183 to investigate the residue’s effect on various steps in the transcription initiation process using the E. coli RNAP. An alanine substitution was created to remove the aromatic while preserving mild hydrophobic properties, a serine substitution introduced hydrophilic properties and aspartate and arginine substitutions introduced negative or positively charged sidechains, respectively. We tested these mutants in a number of in vitro assays to assess their catalytic activity and their capacity to bind DNA and to form holoenzymes (Figure 2).Figure 2.


An aromatic residue switch in enhancer-dependent bacterial RNA polymerase controls transcription intermediate complex activity.

Wiesler SC, Weinzierl RO, Buck M - Nucleic Acids Res. (2013)

Catalytic and binding properties (A) βW183 mutants are defective in nucleic acid binding and holoenzyme formation in comparison with wild-type RNAP (wt). The catalytic defects of the variants can in part be explained by the defects in scaffold binding and holoenzyme formation. The assays were performed as titrations at three different RNAP concentrations (25 nM, 50 nM, 100 nM). The activity for each mutant was calculated relative to the WT activity. The data represent average activities, and the error bars represent standard deviations. For each reaction, 33 nM of α32P-GTP was used. The GTP turnover into RNA achieved by the WT in a minimal scaffold assay (7 nt RNA) was 6 nM (25 nM RNAP), 9 nM (50 nM RNAP) or 17 nM (100 nM RNAP), respectively. (B) Schematic of the minimal scaffold assay measuring RNA elongation (minimal scaffold assay) and pyrophosphorolysis (PPi assay) activities. For minimal scaffold binding assays, the minimal scaffold was 32P-labelled, incubated with RNAP and complexes were separated on a native gel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675486&req=5

gkt271-F2: Catalytic and binding properties (A) βW183 mutants are defective in nucleic acid binding and holoenzyme formation in comparison with wild-type RNAP (wt). The catalytic defects of the variants can in part be explained by the defects in scaffold binding and holoenzyme formation. The assays were performed as titrations at three different RNAP concentrations (25 nM, 50 nM, 100 nM). The activity for each mutant was calculated relative to the WT activity. The data represent average activities, and the error bars represent standard deviations. For each reaction, 33 nM of α32P-GTP was used. The GTP turnover into RNA achieved by the WT in a minimal scaffold assay (7 nt RNA) was 6 nM (25 nM RNAP), 9 nM (50 nM RNAP) or 17 nM (100 nM RNAP), respectively. (B) Schematic of the minimal scaffold assay measuring RNA elongation (minimal scaffold assay) and pyrophosphorolysis (PPi assay) activities. For minimal scaffold binding assays, the minimal scaffold was 32P-labelled, incubated with RNAP and complexes were separated on a native gel.
Mentions: Single residue substitutions were created at position βW183 to investigate the residue’s effect on various steps in the transcription initiation process using the E. coli RNAP. An alanine substitution was created to remove the aromatic while preserving mild hydrophobic properties, a serine substitution introduced hydrophilic properties and aspartate and arginine substitutions introduced negative or positively charged sidechains, respectively. We tested these mutants in a number of in vitro assays to assess their catalytic activity and their capacity to bind DNA and to form holoenzymes (Figure 2).Figure 2.

Bottom Line: A single tryptophan is ideally positioned to directly engage with the flipped out base of the non-template strand at the +1 site.Evidence suggests that this tryptophan (i) is involved in either forward translocation or DNA scrunching and (ii) in σ(54)-regulated promoters limits the transcription activity of at least one intermediate complex (RPi) before the formation of a fully functional RPo.Limiting RPi activity may be important in preventing the premature synthesis of abortive transcripts, suggesting its involvement in a general mechanism driving the RPi to RPo transition for transcription initiation.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK. s.wiesler@imperial.ac.uk

ABSTRACT
The formation of the open promoter complex (RPo) in which the melted DNA containing the transcription start site is located at the RNA polymerase (RNAP) catalytic centre is an obligatory step in the transcription of DNA into RNA catalyzed by RNAP. In the RPo, an extensive network of interactions is established between DNA, RNAP and the σ-factor and the formation of functional RPo occurs via a series of transcriptional intermediates (collectively 'RPi'). A single tryptophan is ideally positioned to directly engage with the flipped out base of the non-template strand at the +1 site. Evidence suggests that this tryptophan (i) is involved in either forward translocation or DNA scrunching and (ii) in σ(54)-regulated promoters limits the transcription activity of at least one intermediate complex (RPi) before the formation of a fully functional RPo. Limiting RPi activity may be important in preventing the premature synthesis of abortive transcripts, suggesting its involvement in a general mechanism driving the RPi to RPo transition for transcription initiation.

Show MeSH