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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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Promoter-binding properties. βW183 mutants are mildly defective in RPc formation, which can be explained by their general defects in holoenzyme formation and DNA binding. Although RPi formation is not affected, the mutants display additive defects in RPo formation. For RPc and RPo, the bars reflect the amount of complex that was formed relative to WT. For RPi, the bars reflect the fraction of RPc that has been converted to RPi for each variant (also see Supplementary Figure S1).
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gkt271-F3: Promoter-binding properties. βW183 mutants are mildly defective in RPc formation, which can be explained by their general defects in holoenzyme formation and DNA binding. Although RPi formation is not affected, the mutants display additive defects in RPo formation. For RPc and RPo, the bars reflect the amount of complex that was formed relative to WT. For RPi, the bars reflect the fraction of RPc that has been converted to RPi for each variant (also see Supplementary Figure S1).

Mentions: The failure to properly engage with nucleic acids and σ54 is directly reflected in the mutants’ modestly compromised ability to form RPcs that reached 60–95% of WT levels (Figure 3). With Eσ54, it is possible to stably form an intermediate complex by the binding of its cognate activator to RPc to form RPi. The RPi can be trapped by adding the nucleotide analogue ADP-AlF, which mimics the conformation of the nucleotide just prior to ATP hydrolysis (26). Formation of the RPi with Eσ54 by adding PspF and ADP-AlF to the RPcs were not affected, and for all RNAP variants comparable fractions (40–45%) of the RPc shifted into the RPi (Figure 3). When allowing the activator-driven ATP hydrolysis reaction to proceed and RPos to form, the defects became more pronounced with the mutants forming 20–60% RPos in comparison to WT (Figure 3). This suggested that, in addition to their defects in binding DNA and σ54, the mutants are also impaired in their capacity to form RPos with the two effects being additive. A 50% reduction in the amount of mutant as compared with WT RPos was demonstrated by forming RPos, followed by adding heparin as a competitor and the removal of samples at regular time intervals to estimate remaining RPo levels. The stability of the complexes formed towards heparin did not seem to be affected by the mutations (Supplementary Figure S1A, right). The presence of the initiating dinucleotide UpG did not significantly stabilize the RPos, thus ruling out the possibility that the defects in RPo stability occur at the level of base pairing to the template strand (Supplementary Figure S1A, left).Figure 3.


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)

Promoter-binding properties. βW183 mutants are mildly defective in RPc formation, which can be explained by their general defects in holoenzyme formation and DNA binding. Although RPi formation is not affected, the mutants display additive defects in RPo formation. For RPc and RPo, the bars reflect the amount of complex that was formed relative to WT. For RPi, the bars reflect the fraction of RPc that has been converted to RPi for each variant (also see Supplementary Figure S1).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675486&req=5

gkt271-F3: Promoter-binding properties. βW183 mutants are mildly defective in RPc formation, which can be explained by their general defects in holoenzyme formation and DNA binding. Although RPi formation is not affected, the mutants display additive defects in RPo formation. For RPc and RPo, the bars reflect the amount of complex that was formed relative to WT. For RPi, the bars reflect the fraction of RPc that has been converted to RPi for each variant (also see Supplementary Figure S1).
Mentions: The failure to properly engage with nucleic acids and σ54 is directly reflected in the mutants’ modestly compromised ability to form RPcs that reached 60–95% of WT levels (Figure 3). With Eσ54, it is possible to stably form an intermediate complex by the binding of its cognate activator to RPc to form RPi. The RPi can be trapped by adding the nucleotide analogue ADP-AlF, which mimics the conformation of the nucleotide just prior to ATP hydrolysis (26). Formation of the RPi with Eσ54 by adding PspF and ADP-AlF to the RPcs were not affected, and for all RNAP variants comparable fractions (40–45%) of the RPc shifted into the RPi (Figure 3). When allowing the activator-driven ATP hydrolysis reaction to proceed and RPos to form, the defects became more pronounced with the mutants forming 20–60% RPos in comparison to WT (Figure 3). This suggested that, in addition to their defects in binding DNA and σ54, the mutants are also impaired in their capacity to form RPos with the two effects being additive. A 50% reduction in the amount of mutant as compared with WT RPos was demonstrated by forming RPos, followed by adding heparin as a competitor and the removal of samples at regular time intervals to estimate remaining RPo levels. The stability of the complexes formed towards heparin did not seem to be affected by the mutations (Supplementary Figure S1A, right). The presence of the initiating dinucleotide UpG did not significantly stabilize the RPos, thus ruling out the possibility that the defects in RPo stability occur at the level of base pairing to the template strand (Supplementary Figure S1A, left).Figure 3.

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