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Structure of a bacterial RNA polymerase holoenzyme open promoter complex.

Bae B, Feklistov A, Lass-Napiorkowska A, Landick R, Darst SA - Elife (2015)

Bottom Line: We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble.The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates.The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Biophysics, The Rockefeller University, New York, United States.

ABSTRACT
Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σ(A) dissociation.

No MeSH data available.


Related in: MedlinePlus

RPo dissociation data.(Left) Representative time trace of fluorescence decay after rapid mixing of pre-formed Eco RPo (with wild-type σ70) into 1.1 M NaCl (Gries et al., 2010). The solid line illustrates the non-linear regression fit to a single-exponential model. (Right) Representative dissociation curves for holoenzymes containing wild-type σ70 and W433A and Y394A substitutions.DOI:http://dx.doi.org/10.7554/eLife.08504.016
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fig5s1: RPo dissociation data.(Left) Representative time trace of fluorescence decay after rapid mixing of pre-formed Eco RPo (with wild-type σ70) into 1.1 M NaCl (Gries et al., 2010). The solid line illustrates the non-linear regression fit to a single-exponential model. (Right) Representative dissociation curves for holoenzymes containing wild-type σ70 and W433A and Y394A substitutions.DOI:http://dx.doi.org/10.7554/eLife.08504.016

Mentions: W256 appears to make the primary contribution to maintaining the ds/ss junction at the upstream edge of the transcription bubble (Figure 3A), suggesting that this residue may play an important role in preventing transcription bubble collapse and dissociation of RPo. To probe the roles of both σ70 W433 and Y394 in maintaining RPo stability, we rapidly destabilized preformed RPo with 1.1 M NaCl (Gries et al., 2010) and followed the loss of RPo by monitoring the decay of fluorescence intensity with time (Figure 5—figure supplement 1). The dissociation curves are complex, reflecting the detection of a short lived intermediate (expected under these conditions) (Gries et al., 2010) by this assay. Although a full analysis is beyond the scope of this study, the overall apparent rate of RPo decay was determined from single-exponential fits of the decay curves. The σ70 W433A and the Y394A variants both gave a ∼fourfold higher rate of RPo dissociation under high salt conditions than did wild-type σ70 (Figure 5D, Figure 5—figure supplement 1).


Structure of a bacterial RNA polymerase holoenzyme open promoter complex.

Bae B, Feklistov A, Lass-Napiorkowska A, Landick R, Darst SA - Elife (2015)

RPo dissociation data.(Left) Representative time trace of fluorescence decay after rapid mixing of pre-formed Eco RPo (with wild-type σ70) into 1.1 M NaCl (Gries et al., 2010). The solid line illustrates the non-linear regression fit to a single-exponential model. (Right) Representative dissociation curves for holoenzymes containing wild-type σ70 and W433A and Y394A substitutions.DOI:http://dx.doi.org/10.7554/eLife.08504.016
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4593229&req=5

fig5s1: RPo dissociation data.(Left) Representative time trace of fluorescence decay after rapid mixing of pre-formed Eco RPo (with wild-type σ70) into 1.1 M NaCl (Gries et al., 2010). The solid line illustrates the non-linear regression fit to a single-exponential model. (Right) Representative dissociation curves for holoenzymes containing wild-type σ70 and W433A and Y394A substitutions.DOI:http://dx.doi.org/10.7554/eLife.08504.016
Mentions: W256 appears to make the primary contribution to maintaining the ds/ss junction at the upstream edge of the transcription bubble (Figure 3A), suggesting that this residue may play an important role in preventing transcription bubble collapse and dissociation of RPo. To probe the roles of both σ70 W433 and Y394 in maintaining RPo stability, we rapidly destabilized preformed RPo with 1.1 M NaCl (Gries et al., 2010) and followed the loss of RPo by monitoring the decay of fluorescence intensity with time (Figure 5—figure supplement 1). The dissociation curves are complex, reflecting the detection of a short lived intermediate (expected under these conditions) (Gries et al., 2010) by this assay. Although a full analysis is beyond the scope of this study, the overall apparent rate of RPo decay was determined from single-exponential fits of the decay curves. The σ70 W433A and the Y394A variants both gave a ∼fourfold higher rate of RPo dissociation under high salt conditions than did wild-type σ70 (Figure 5D, Figure 5—figure supplement 1).

Bottom Line: We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble.The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates.The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Biophysics, The Rockefeller University, New York, United States.

ABSTRACT
Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σ(A) dissociation.

No MeSH data available.


Related in: MedlinePlus