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CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex.

Bae B, Chen J, Davis E, Leon K, Darst SA, Campbell EA - Elife (2015)

Bottom Line: The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble.We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex.Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD.

View Article: PubMed Central - PubMed

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

ABSTRACT
A key point to regulate gene expression is at transcription initiation, and activators play a major role. CarD, an essential activator in Mycobacterium tuberculosis, is found in many bacteria, including Thermus species, but absent in Escherichia coli. To delineate the molecular mechanism of CarD, we determined crystal structures of Thermus transcription initiation complexes containing CarD. The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble. We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD.

No MeSH data available.


Related in: MedlinePlus

CarD/β1-lobe structure.(Top) View of the CarD/RPo structure, similar to Figure 1B (Right) except the RNAP β1-lobe and CarD are shown as backbone ribbons without surfaces. (Bottom) The CarD/β1-lobe structure (2.4 Å-resolution, Table 1) shown in the orientation corresponding to the top view.DOI:http://dx.doi.org/10.7554/eLife.08505.007
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fig1s3: CarD/β1-lobe structure.(Top) View of the CarD/RPo structure, similar to Figure 1B (Right) except the RNAP β1-lobe and CarD are shown as backbone ribbons without surfaces. (Bottom) The CarD/β1-lobe structure (2.4 Å-resolution, Table 1) shown in the orientation corresponding to the top view.DOI:http://dx.doi.org/10.7554/eLife.08505.007

Mentions: Crystals of CarD transcription activation complexes were prepared by soaking Tth CarD into Taq Δ1.1σA-holoenzyme/us-fork (−12 bp) or full RPo crystals (Bae et al., 2015). Analysis of the diffraction data indicated high occupancy of one CarD molecule bound to each of two RNAP/promoter complexes in the asymmetric unit of the crystal lattice (Figure 1—figure supplement 2). Docking CarD onto the RNAP was facilitated by a high-resolution crystal structure of a Tth CarD/Taq β1-lobe complex (2.4 Å-resolution, Table 2, Figure 1—figure supplement 3, Figure 1—figure supplement 4). The structures of CarD transcription activation complexes were refined to 4.4 and 4.3 Å-resolution, respectively (Table 2, Figure 1—figure supplement 5). The protein/protein and protein/nucleic acid interactions were essentially identical among all of the four crystallographically independent complexes, so the more complete and higher resolution CarD/RPo structure (Figure 1A,B, Figure 1—figure supplement 5, Table 2) is described here. Although the CarD bound to one RPo in the crystallographic asymmetric unit made crystal-packing interactions with a symmetry-related CarD, the CarD bound to the second RPo did not participate in any crystal-packing interactions (Figure 1—figure supplement 2), indicating the architecture and interactions observed here are unlikely to be influenced by crystal packing interactions and likely represent the functional activation complex in solution.10.7554/eLife.08505.011Table 2.


CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex.

Bae B, Chen J, Davis E, Leon K, Darst SA, Campbell EA - Elife (2015)

CarD/β1-lobe structure.(Top) View of the CarD/RPo structure, similar to Figure 1B (Right) except the RNAP β1-lobe and CarD are shown as backbone ribbons without surfaces. (Bottom) The CarD/β1-lobe structure (2.4 Å-resolution, Table 1) shown in the orientation corresponding to the top view.DOI:http://dx.doi.org/10.7554/eLife.08505.007
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Related In: Results  -  Collection

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fig1s3: CarD/β1-lobe structure.(Top) View of the CarD/RPo structure, similar to Figure 1B (Right) except the RNAP β1-lobe and CarD are shown as backbone ribbons without surfaces. (Bottom) The CarD/β1-lobe structure (2.4 Å-resolution, Table 1) shown in the orientation corresponding to the top view.DOI:http://dx.doi.org/10.7554/eLife.08505.007
Mentions: Crystals of CarD transcription activation complexes were prepared by soaking Tth CarD into Taq Δ1.1σA-holoenzyme/us-fork (−12 bp) or full RPo crystals (Bae et al., 2015). Analysis of the diffraction data indicated high occupancy of one CarD molecule bound to each of two RNAP/promoter complexes in the asymmetric unit of the crystal lattice (Figure 1—figure supplement 2). Docking CarD onto the RNAP was facilitated by a high-resolution crystal structure of a Tth CarD/Taq β1-lobe complex (2.4 Å-resolution, Table 2, Figure 1—figure supplement 3, Figure 1—figure supplement 4). The structures of CarD transcription activation complexes were refined to 4.4 and 4.3 Å-resolution, respectively (Table 2, Figure 1—figure supplement 5). The protein/protein and protein/nucleic acid interactions were essentially identical among all of the four crystallographically independent complexes, so the more complete and higher resolution CarD/RPo structure (Figure 1A,B, Figure 1—figure supplement 5, Table 2) is described here. Although the CarD bound to one RPo in the crystallographic asymmetric unit made crystal-packing interactions with a symmetry-related CarD, the CarD bound to the second RPo did not participate in any crystal-packing interactions (Figure 1—figure supplement 2), indicating the architecture and interactions observed here are unlikely to be influenced by crystal packing interactions and likely represent the functional activation complex in solution.10.7554/eLife.08505.011Table 2.

Bottom Line: The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble.We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex.Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD.

View Article: PubMed Central - PubMed

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

ABSTRACT
A key point to regulate gene expression is at transcription initiation, and activators play a major role. CarD, an essential activator in Mycobacterium tuberculosis, is found in many bacteria, including Thermus species, but absent in Escherichia coli. To delineate the molecular mechanism of CarD, we determined crystal structures of Thermus transcription initiation complexes containing CarD. The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble. We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD.

No MeSH data available.


Related in: MedlinePlus