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

Brian Bae,James Chen,Katherine Leon,Elizabeth Davis,Seth A Darst,Elizabeth A Campbell

doi:10.7554/elife.08505

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.

Highlights

Decades of research using Escherichia coli (Eco) as a model system inform most of our understanding of how bacteria control transcription initiation
The CarD bound to one RNAP holoenzyme open promoter complex (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
CarD is an essential transcription activator in Mycobacterium tuberculosis (Mtb) that is widely distributed among bacterial species, including Thermus species but not found in Eco (Stallings et al, 2009; Srivastava et al, 2013; Table 1)

Summary

Introduction

Decades of research using Escherichia coli (Eco) as a model system inform most of our understanding of how bacteria control transcription initiation. Dissociable promoter specificity subunits, σ factors, direct the catalytic core of the RNA polymerase (RNAP) to promoter DNA sites and play a key role in unwinding the DNA duplex to create the transcription bubble in the RNAP holoenzyme open promoter complex (RPo) (Feklistov et al, 2014). Activators can accelerate initiation by stabilizing the initial RNAP/promoter complex, by stimulating the isomerization of the initial RNAP/promoter complex to RPo (i.e., unwinding the duplex DNA to form the transcription bubble), or both (Li et al, 1997; Roy et al, 1998). CarD, first identified as a regulator of ribosomal RNA (rRNA) transcription in Mycobacterium tuberculosis (Mtb), is a transcriptional activator widely distributed among bacterial species, including Thermus species (Stallings et al, 2009; Srivastava et al, 2013), but is absent in Eco (Table 1). A deeper understanding of the CarD functional mechanism and its role in the Mtb transcription program is warranted

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