Limits...
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

Complete gels for the abortive initiation assays shown in Figure 6B.DOI:http://dx.doi.org/10.7554/eLife.08505.022
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4593161&req=5

fig6s1: Complete gels for the abortive initiation assays shown in Figure 6B.DOI:http://dx.doi.org/10.7554/eLife.08505.022

Mentions: Clearly, dissociation of RPo and transcription bubble collapse (rewinding) are closely linked. We hypothesized that CarD may increase the lifetime of RPo by preventing transcription bubble collapse. To test this hypothesis, we determined the effect of CarD on the lifetime of promoter complexes on a synthetic promoter template based on the 23S sequence and compared it with the same synthetic template but with a non-complementary transcription bubble (from −11 to +2) unable to collapse (Figure 6A). On the duplex template (23S_DS), CarD increased the t1/2 more than fivefold (Figure 6B, Figure 6—figure supplement 1). On the bubble template (Figure 6A, 23S_Bub) in the absence of CarD, the t1/2 was also increased more than fivefold, indicating that the relatively short lifetime of Tth RPo on the 23S promoter is due largely to bubble collapse (Figure 6B). Addition of CarD to the bubble template had no effect on the level of transcription and did not affect RPo lifetime (Figure 6B). We thus conclude that a primary function of Tth CarD, like Mtb CarD (Davis et al., 2015), is to stabilize RPo by preventing collapse of the transcription bubble.10.7554/eLife.08505.021Figure 6.CarD increases the lifetime of Thermus RPo by preventing transcription bubble collapse.


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)

Complete gels for the abortive initiation assays shown in Figure 6B.DOI:http://dx.doi.org/10.7554/eLife.08505.022
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4593161&req=5

fig6s1: Complete gels for the abortive initiation assays shown in Figure 6B.DOI:http://dx.doi.org/10.7554/eLife.08505.022
Mentions: Clearly, dissociation of RPo and transcription bubble collapse (rewinding) are closely linked. We hypothesized that CarD may increase the lifetime of RPo by preventing transcription bubble collapse. To test this hypothesis, we determined the effect of CarD on the lifetime of promoter complexes on a synthetic promoter template based on the 23S sequence and compared it with the same synthetic template but with a non-complementary transcription bubble (from −11 to +2) unable to collapse (Figure 6A). On the duplex template (23S_DS), CarD increased the t1/2 more than fivefold (Figure 6B, Figure 6—figure supplement 1). On the bubble template (Figure 6A, 23S_Bub) in the absence of CarD, the t1/2 was also increased more than fivefold, indicating that the relatively short lifetime of Tth RPo on the 23S promoter is due largely to bubble collapse (Figure 6B). Addition of CarD to the bubble template had no effect on the level of transcription and did not affect RPo lifetime (Figure 6B). We thus conclude that a primary function of Tth CarD, like Mtb CarD (Davis et al., 2015), is to stabilize RPo by preventing collapse of the transcription bubble.10.7554/eLife.08505.021Figure 6.CarD increases the lifetime of Thermus RPo by preventing transcription bubble collapse.

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