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

Area of Science:

• Molecular biology
• Structural biology
• Biochemistry

Background:

• Gene expression regulation is critical, with transcription initiation being a key control point.
• Activators like CarD are essential for transcription, particularly in bacteria like Mycobacterium tuberculosis and Thermus species.
• Escherichia coli RNAP forms stable promoter complexes, unlike many other bacterial RNAPs that require activators like CarD.

Purpose of the Study:

• To elucidate the molecular mechanism of CarD's function in transcription initiation.
• To determine the structural basis of CarD's interaction with transcription initiation complexes.

Main Methods:

• X-ray crystallography was used to determine the structures of Thermus transcription initiation complexes containing CarD.
• Structural analysis focused on CarD's interaction with the DNA topology at the transcription bubble.
• Functional assays confirmed the relevance of the observed structures.

Main Results:

• Crystal structures revealed CarD interacts with the DNA junction of the transcription bubble.
• A conserved CarD tryptophan residue acts as a minor groove wedge, stabilizing the transcription bubble.
• These interactions prevent the collapse of the transcription bubble, stabilizing the initiation complex.

Conclusions:

• CarD stabilizes transcription initiation complexes by interacting with unique DNA structures.
• The minor groove wedge mechanism is crucial for CarD's function in stabilizing the transcription bubble.
• The findings explain the necessity of CarD in bacteria with inherently unstable promoter complexes.