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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

Data and model quality for us-fork (−12 bp) and RPo complexes.Plots relating data quality with model quality at 4.0 Å-resolution) using the Pearson correlation coefficient (CC) analysis described by Karplus and Diederichs (2012). CC1/2 (red squares) was determined from the unmerged diffraction data randomly divided in half. Since CC1/2 underestimates the information content of the data (since it's calculated by dividing the dataset in half), CC* was calculated from an analytical relation to estimate the information content of the full data (Karplus and Diederichs, 2012). CC* provides a statistic that assesses data quality as well and also allows direct comparison of crystallographic model quality and data quality on the same scale through CCwork and CCfree, the standard and cross-validated correlations of the experimental intensities with the intensities calculated from the refined model. A CCwork/CCfree smaller than CC* indicates that the model does not account for all of the signal in the data, meaning it is not overfit. Plotted also are the standard <I>/σI for the diffraction data, as well as the Rwork/Rfree for the refined models. (Left) Data for Taq EΔ1.1σA/us-fork (−12 bp) at 4.0 Å-resolution. (Right) Data for Taq EΔ1.1σA RPo (with 4-nt RNA primer) at 4.0 Å-resolution.DOI:http://dx.doi.org/10.7554/eLife.08504.005
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fig1s2: Data and model quality for us-fork (−12 bp) and RPo complexes.Plots relating data quality with model quality at 4.0 Å-resolution) using the Pearson correlation coefficient (CC) analysis described by Karplus and Diederichs (2012). CC1/2 (red squares) was determined from the unmerged diffraction data randomly divided in half. Since CC1/2 underestimates the information content of the data (since it's calculated by dividing the dataset in half), CC* was calculated from an analytical relation to estimate the information content of the full data (Karplus and Diederichs, 2012). CC* provides a statistic that assesses data quality as well and also allows direct comparison of crystallographic model quality and data quality on the same scale through CCwork and CCfree, the standard and cross-validated correlations of the experimental intensities with the intensities calculated from the refined model. A CCwork/CCfree smaller than CC* indicates that the model does not account for all of the signal in the data, meaning it is not overfit. Plotted also are the standard <I>/σI for the diffraction data, as well as the Rwork/Rfree for the refined models. (Left) Data for Taq EΔ1.1σA/us-fork (−12 bp) at 4.0 Å-resolution. (Right) Data for Taq EΔ1.1σA RPo (with 4-nt RNA primer) at 4.0 Å-resolution.DOI:http://dx.doi.org/10.7554/eLife.08504.005

Mentions: Structures of Escherichia coli (Eco) transcription initiation complexes containing a complete transcription bubble delineated the overall architecture of the full bubble, but the low resolution of the analyses (between 5.5 and 6 Å resolution) prevented a detailed description of protein/DNA interactions (Zuo and Steitz, 2015). Here, we determine crystal structures of Taq EσA bound to an us-fork promoter fragment, as well as a complete RPo (Figure 1, Figure 1—figure supplement 1), refined using diffraction data extending to 4.00 and 4.14 Å-resolution, respectively (Table 1, Figure 1—figure supplement 2), allowing visualization of key features that stabilize the upstream edge of the transcription bubble. The results also reveal functionally relevant holoenzyme interactions with duplex DNA just upstream of the −10 element and potential protein/DNA interactions that direct the DNA template strand (t-strand) into the RNAP active site. Addition of an RNA primer to yield a 4-bp post-translocated RNA:DNA hybrid mimics RPITC at the point where steric clash initiates abortive initiation and σA dissociation (Murakami et al., 2002a; Kulbachinskiy and Mustaev, 2006).10.7554/eLife.08504.003Figure 1.Structure of RPo.


Structure of a bacterial RNA polymerase holoenzyme open promoter complex.

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

Data and model quality for us-fork (−12 bp) and RPo complexes.Plots relating data quality with model quality at 4.0 Å-resolution) using the Pearson correlation coefficient (CC) analysis described by Karplus and Diederichs (2012). CC1/2 (red squares) was determined from the unmerged diffraction data randomly divided in half. Since CC1/2 underestimates the information content of the data (since it's calculated by dividing the dataset in half), CC* was calculated from an analytical relation to estimate the information content of the full data (Karplus and Diederichs, 2012). CC* provides a statistic that assesses data quality as well and also allows direct comparison of crystallographic model quality and data quality on the same scale through CCwork and CCfree, the standard and cross-validated correlations of the experimental intensities with the intensities calculated from the refined model. A CCwork/CCfree smaller than CC* indicates that the model does not account for all of the signal in the data, meaning it is not overfit. Plotted also are the standard <I>/σI for the diffraction data, as well as the Rwork/Rfree for the refined models. (Left) Data for Taq EΔ1.1σA/us-fork (−12 bp) at 4.0 Å-resolution. (Right) Data for Taq EΔ1.1σA RPo (with 4-nt RNA primer) at 4.0 Å-resolution.DOI:http://dx.doi.org/10.7554/eLife.08504.005
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fig1s2: Data and model quality for us-fork (−12 bp) and RPo complexes.Plots relating data quality with model quality at 4.0 Å-resolution) using the Pearson correlation coefficient (CC) analysis described by Karplus and Diederichs (2012). CC1/2 (red squares) was determined from the unmerged diffraction data randomly divided in half. Since CC1/2 underestimates the information content of the data (since it's calculated by dividing the dataset in half), CC* was calculated from an analytical relation to estimate the information content of the full data (Karplus and Diederichs, 2012). CC* provides a statistic that assesses data quality as well and also allows direct comparison of crystallographic model quality and data quality on the same scale through CCwork and CCfree, the standard and cross-validated correlations of the experimental intensities with the intensities calculated from the refined model. A CCwork/CCfree smaller than CC* indicates that the model does not account for all of the signal in the data, meaning it is not overfit. Plotted also are the standard <I>/σI for the diffraction data, as well as the Rwork/Rfree for the refined models. (Left) Data for Taq EΔ1.1σA/us-fork (−12 bp) at 4.0 Å-resolution. (Right) Data for Taq EΔ1.1σA RPo (with 4-nt RNA primer) at 4.0 Å-resolution.DOI:http://dx.doi.org/10.7554/eLife.08504.005
Mentions: Structures of Escherichia coli (Eco) transcription initiation complexes containing a complete transcription bubble delineated the overall architecture of the full bubble, but the low resolution of the analyses (between 5.5 and 6 Å resolution) prevented a detailed description of protein/DNA interactions (Zuo and Steitz, 2015). Here, we determine crystal structures of Taq EσA bound to an us-fork promoter fragment, as well as a complete RPo (Figure 1, Figure 1—figure supplement 1), refined using diffraction data extending to 4.00 and 4.14 Å-resolution, respectively (Table 1, Figure 1—figure supplement 2), allowing visualization of key features that stabilize the upstream edge of the transcription bubble. The results also reveal functionally relevant holoenzyme interactions with duplex DNA just upstream of the −10 element and potential protein/DNA interactions that direct the DNA template strand (t-strand) into the RNAP active site. Addition of an RNA primer to yield a 4-bp post-translocated RNA:DNA hybrid mimics RPITC at the point where steric clash initiates abortive initiation and σA dissociation (Murakami et al., 2002a; Kulbachinskiy and Mustaev, 2006).10.7554/eLife.08504.003Figure 1.Structure of RPo.

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