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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

Structural role of the σ3.2-loop.(Left) Overall view of RPo structure, colored as in Figure 1 except σA is orange. The RNAP β and β′ subunits are transparent to reveal the RNAP active site Mg2+ (yellow sphere) and the nucleic acids held inside the RNAP active site channel. The ss nt-strand DNA is omitted for clarity. The boxed area is magnified on the right. (Right) Magnified view showing a cross-section of the RNAP active site channel. For clarity, the RNAP β, β′, and  domains are shown mostly as outlined shapes, with β transparent. The ss t-strand DNA (−11 to −4) is directed towards the RNAP active site through a tunnel between the σ3.2-loop and the β1-lobe. The 4-nt RNA transcript (−3 to +1) contacts the distal tip of the σ3.2-loop. Further elongation of the RNA would require displacement of the σ3.2-loop.DOI:http://dx.doi.org/10.7554/eLife.08504.017
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fig6: Structural role of the σ3.2-loop.(Left) Overall view of RPo structure, colored as in Figure 1 except σA is orange. The RNAP β and β′ subunits are transparent to reveal the RNAP active site Mg2+ (yellow sphere) and the nucleic acids held inside the RNAP active site channel. The ss nt-strand DNA is omitted for clarity. The boxed area is magnified on the right. (Right) Magnified view showing a cross-section of the RNAP active site channel. For clarity, the RNAP β, β′, and domains are shown mostly as outlined shapes, with β transparent. The ss t-strand DNA (−11 to −4) is directed towards the RNAP active site through a tunnel between the σ3.2-loop and the β1-lobe. The 4-nt RNA transcript (−3 to +1) contacts the distal tip of the σ3.2-loop. Further elongation of the RNA would require displacement of the σ3.2-loop.DOI:http://dx.doi.org/10.7554/eLife.08504.017

Mentions: At the point of melting, a ∼90° turn of the t-strand backbone (between −12 and −11) may be effected by electrostatic interactions between conserved basic residues of (R220; Figure 1—figure supplement 3; Table 3) and (R288, R291) and four t-strand backbone phosphates in a row (−13, −12, −11, −10) encompassing the turn (Figure 3A). Strong simulated annealing omit 2Fo − Fc density is associated wth R288, confirming its role in interacting with the −13(t) phosphate (Figure 3E). The R220 and R291 give weaker difference density so their role in interacting with the −12(t) and −11(t) phosphate groups is tentative. The turn directs the t-strand away from the nt-strand and towards the RNAP active site (Figure 3A). The ss t-strand DNA from −9 to −5 is guided towards the RNAP active site through a tunnel formed between the RNAP β1-lobe (called the protrusion in eukaryotic RNAP II; Cramer et al., 2001) and the σ3.2-loop (also referred to as the σ-finger), an extended linker that loops into and out of the RNAP active-site channel (Murakami et al., 2002a; Zhang et al., 2012), connecting the σ3 and σ4 domains (Figure 6).10.7554/eLife.08504.017Figure 6.Structural role of the σ3.2-loop.


Structure of a bacterial RNA polymerase holoenzyme open promoter complex.

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

Structural role of the σ3.2-loop.(Left) Overall view of RPo structure, colored as in Figure 1 except σA is orange. The RNAP β and β′ subunits are transparent to reveal the RNAP active site Mg2+ (yellow sphere) and the nucleic acids held inside the RNAP active site channel. The ss nt-strand DNA is omitted for clarity. The boxed area is magnified on the right. (Right) Magnified view showing a cross-section of the RNAP active site channel. For clarity, the RNAP β, β′, and  domains are shown mostly as outlined shapes, with β transparent. The ss t-strand DNA (−11 to −4) is directed towards the RNAP active site through a tunnel between the σ3.2-loop and the β1-lobe. The 4-nt RNA transcript (−3 to +1) contacts the distal tip of the σ3.2-loop. Further elongation of the RNA would require displacement of the σ3.2-loop.DOI:http://dx.doi.org/10.7554/eLife.08504.017
© Copyright Policy
Related In: Results  -  Collection

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fig6: Structural role of the σ3.2-loop.(Left) Overall view of RPo structure, colored as in Figure 1 except σA is orange. The RNAP β and β′ subunits are transparent to reveal the RNAP active site Mg2+ (yellow sphere) and the nucleic acids held inside the RNAP active site channel. The ss nt-strand DNA is omitted for clarity. The boxed area is magnified on the right. (Right) Magnified view showing a cross-section of the RNAP active site channel. For clarity, the RNAP β, β′, and domains are shown mostly as outlined shapes, with β transparent. The ss t-strand DNA (−11 to −4) is directed towards the RNAP active site through a tunnel between the σ3.2-loop and the β1-lobe. The 4-nt RNA transcript (−3 to +1) contacts the distal tip of the σ3.2-loop. Further elongation of the RNA would require displacement of the σ3.2-loop.DOI:http://dx.doi.org/10.7554/eLife.08504.017
Mentions: At the point of melting, a ∼90° turn of the t-strand backbone (between −12 and −11) may be effected by electrostatic interactions between conserved basic residues of (R220; Figure 1—figure supplement 3; Table 3) and (R288, R291) and four t-strand backbone phosphates in a row (−13, −12, −11, −10) encompassing the turn (Figure 3A). Strong simulated annealing omit 2Fo − Fc density is associated wth R288, confirming its role in interacting with the −13(t) phosphate (Figure 3E). The R220 and R291 give weaker difference density so their role in interacting with the −12(t) and −11(t) phosphate groups is tentative. The turn directs the t-strand away from the nt-strand and towards the RNAP active site (Figure 3A). The ss t-strand DNA from −9 to −5 is guided towards the RNAP active site through a tunnel formed between the RNAP β1-lobe (called the protrusion in eukaryotic RNAP II; Cramer et al., 2001) and the σ3.2-loop (also referred to as the σ-finger), an extended linker that loops into and out of the RNAP active-site channel (Murakami et al., 2002a; Zhang et al., 2012), connecting the σ3 and σ4 domains (Figure 6).10.7554/eLife.08504.017Figure 6.Structural role of the σ3.2-loop.

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