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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: A key point to regulate gene expression is at transcription initiation, and activators play a major role.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

Alignment of CarD homologs found in bacteria from 11 diverse phyla/groups.The CarD sequences shown are from the following organisms chosen to represent the preceding phylum/group: Deinococcus–Thermus-Tth HB8, Actinobacteria–Mtb, Acidobacteria–Candidatus Solibacter usitatus, α-Proteobacteria–Rickettsia belli, Aquificae–Desulfurobacterium thermolithotrophum, Chlamydae–Chlamydae trachomatis, Cyanobacteria–Mastigocoleus testarum, δ-Proteobacteria–Desulfobulbus propionicus, Firmicutes–Bacillus cereus, Spirochaetes–Treponema pallidum and Thermodesulfobacteria–Thermodesulfatator atlanticus. Alignments were performed using the ClustalW algorithm in MegAlign (DNASTAR). Groups of residues considered homologous (DE), (HKR), (ALMIV), (NQ), (ST), (C), (G) and (P) are shaded blue when occurring in greater than 9/11 sequences. Identical residues occurring in all 11 sequences are shaded black. Histograms above the alignment graphically illustrate residues that are absolutely conserved within each of the 11 sequences and W86 is asterisked. The 100% identical residue is listed immediately below the histogram. A larger alignment of 831 CarD sequences is included (Source code 1).DOI:http://dx.doi.org/10.7554/eLife.08505.013
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fig2s1: Alignment of CarD homologs found in bacteria from 11 diverse phyla/groups.The CarD sequences shown are from the following organisms chosen to represent the preceding phylum/group: Deinococcus–Thermus-Tth HB8, Actinobacteria–Mtb, Acidobacteria–Candidatus Solibacter usitatus, α-Proteobacteria–Rickettsia belli, Aquificae–Desulfurobacterium thermolithotrophum, Chlamydae–Chlamydae trachomatis, Cyanobacteria–Mastigocoleus testarum, δ-Proteobacteria–Desulfobulbus propionicus, Firmicutes–Bacillus cereus, Spirochaetes–Treponema pallidum and Thermodesulfobacteria–Thermodesulfatator atlanticus. Alignments were performed using the ClustalW algorithm in MegAlign (DNASTAR). Groups of residues considered homologous (DE), (HKR), (ALMIV), (NQ), (ST), (C), (G) and (P) are shaded blue when occurring in greater than 9/11 sequences. Identical residues occurring in all 11 sequences are shaded black. Histograms above the alignment graphically illustrate residues that are absolutely conserved within each of the 11 sequences and W86 is asterisked. The 100% identical residue is listed immediately below the histogram. A larger alignment of 831 CarD sequences is included (Source code 1).DOI:http://dx.doi.org/10.7554/eLife.08505.013

Mentions: W86 is conserved among greater than 95% of CarD proteins (Figure 2—figure supplement 1; Source code 1) and was shown to be important for CarD function as an activator (Srivastava et al., 2013). The bulky, hydrophobic planar side chain of W86, located at the N-terminal end of α3, wedges into the splayed minor groove at the upstream edge of the transcription bubble (Figure 2). Despite the relatively low resolution of our analysis (Table 2), CarD-W86 was clearly resolved in electron density maps (Figure 2A). The positioning of CarD-W86 was further supported by an unbiased simulated annealing omit Fo − Fc map calculated from coordinates in which CarD-W86 had been mutated to Ala (Figure 2C).


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)

Alignment of CarD homologs found in bacteria from 11 diverse phyla/groups.The CarD sequences shown are from the following organisms chosen to represent the preceding phylum/group: Deinococcus–Thermus-Tth HB8, Actinobacteria–Mtb, Acidobacteria–Candidatus Solibacter usitatus, α-Proteobacteria–Rickettsia belli, Aquificae–Desulfurobacterium thermolithotrophum, Chlamydae–Chlamydae trachomatis, Cyanobacteria–Mastigocoleus testarum, δ-Proteobacteria–Desulfobulbus propionicus, Firmicutes–Bacillus cereus, Spirochaetes–Treponema pallidum and Thermodesulfobacteria–Thermodesulfatator atlanticus. Alignments were performed using the ClustalW algorithm in MegAlign (DNASTAR). Groups of residues considered homologous (DE), (HKR), (ALMIV), (NQ), (ST), (C), (G) and (P) are shaded blue when occurring in greater than 9/11 sequences. Identical residues occurring in all 11 sequences are shaded black. Histograms above the alignment graphically illustrate residues that are absolutely conserved within each of the 11 sequences and W86 is asterisked. The 100% identical residue is listed immediately below the histogram. A larger alignment of 831 CarD sequences is included (Source code 1).DOI:http://dx.doi.org/10.7554/eLife.08505.013
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4593161&req=5

fig2s1: Alignment of CarD homologs found in bacteria from 11 diverse phyla/groups.The CarD sequences shown are from the following organisms chosen to represent the preceding phylum/group: Deinococcus–Thermus-Tth HB8, Actinobacteria–Mtb, Acidobacteria–Candidatus Solibacter usitatus, α-Proteobacteria–Rickettsia belli, Aquificae–Desulfurobacterium thermolithotrophum, Chlamydae–Chlamydae trachomatis, Cyanobacteria–Mastigocoleus testarum, δ-Proteobacteria–Desulfobulbus propionicus, Firmicutes–Bacillus cereus, Spirochaetes–Treponema pallidum and Thermodesulfobacteria–Thermodesulfatator atlanticus. Alignments were performed using the ClustalW algorithm in MegAlign (DNASTAR). Groups of residues considered homologous (DE), (HKR), (ALMIV), (NQ), (ST), (C), (G) and (P) are shaded blue when occurring in greater than 9/11 sequences. Identical residues occurring in all 11 sequences are shaded black. Histograms above the alignment graphically illustrate residues that are absolutely conserved within each of the 11 sequences and W86 is asterisked. The 100% identical residue is listed immediately below the histogram. A larger alignment of 831 CarD sequences is included (Source code 1).DOI:http://dx.doi.org/10.7554/eLife.08505.013
Mentions: W86 is conserved among greater than 95% of CarD proteins (Figure 2—figure supplement 1; Source code 1) and was shown to be important for CarD function as an activator (Srivastava et al., 2013). The bulky, hydrophobic planar side chain of W86, located at the N-terminal end of α3, wedges into the splayed minor groove at the upstream edge of the transcription bubble (Figure 2). Despite the relatively low resolution of our analysis (Table 2), CarD-W86 was clearly resolved in electron density maps (Figure 2A). The positioning of CarD-W86 was further supported by an unbiased simulated annealing omit Fo − Fc map calculated from coordinates in which CarD-W86 had been mutated to Ala (Figure 2C).

Bottom Line: A key point to regulate gene expression is at transcription initiation, and activators play a major role.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