Structural basis for the bacterial transcription-repair coupling factor/RNA polymerase interaction

Abstract

The transcription-repair coupling factor (TRCF, the product of the mfd gene) is a widely conserved bacterial protein that mediates transcription-coupled DNA repair. TRCF uses its ATP-dependent DNA translocase activity to remove transcription complexes stalled at sites of DNA damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. A protein/protein interaction between TRCF and the β-subunit of RNA polymerase (RNAP) is essential for TRCF function. CarD (also called CdnL), an essential regulator of rRNA transcription in Mycobacterium tuberculosis, shares a homologous RNAP interacting domain with TRCF and also interacts with the RNAP β-subunit. We determined the 2.9-Å resolution X-ray crystal structure of the RNAP interacting domain of TRCF complexed with the RNAP-β1 domain, which harbors the TRCF interaction determinants. The structure reveals details of the TRCF/RNAP protein/protein interface, providing a basis for the design and interpretation of experiments probing TRCF, and by homology CarD, function and interactions with the RNAP.

Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis

“…The N terminus of the CarD protein displays amino acid sequence similarity to that of the RNAP interaction domain (RID) of the TRCF (the product of the mfd gene). We previously reported that Thermus thermophilus CarD interacts with the same region at the N terminus of the RNAP ␤ subunit (the ␤1 domain) as the T. thermophilus TRCF (22,24). Studies of Myxococcus xanthus proteins have reported similar findings (10).…”

“…The X-ray crystal structure of the T. thermophilus TRCF RID complexed with the Thermus aquaticus RNAP ␤1 domain (24) revealed that T. thermophilus TRCF RID R341 forms a hydrogen bond with RNAP ␤1 Q99, as well as polar interactions with RNAP ␤1 E110. The RNAP ␤1 residues I108 and K109 are also central to the protein/protein interface, making extensive van der Waals contacts with the TRCF RID (24).…”

“…The X-ray crystal structure of the T. thermophilus TRCF RID complexed with the Thermus aquaticus RNAP ␤1 domain (24) revealed that T. thermophilus TRCF RID R341 forms a hydrogen bond with RNAP ␤1 Q99, as well as polar interactions with RNAP ␤1 E110. The RNAP ␤1 residues I108 and K109 are also central to the protein/protein interface, making extensive van der Waals contacts with the TRCF RID (24). T. thermophilus TRCF RID R341 and T. aquaticus RNAP ␤1 E110 correspond to Escherichia coli TRCF RID L499 and RNAP ␤1 E119, respectively, which were both previously demonstrated to be important for the interaction between the TRCF and RNAP ␤ in E. coli (6,20).…”

Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

“…S7C) and, consequently, the dynamic equilibrium of TRCF conformations. Thus, the robust TEC release activity of oxidized TRCF-D2:D7, which is greatly impaired in DNA binding, suggests that the initial recognition of a stalled TEC is likely provided through specific protein-protein contacts between the RID and the β-subunit of RNAP (5,18,35) and that protein-DNA contacts play a secondary role.…”

Mfd as a central partner of transcription coupled repair

“…UvrC then nicks the DNA on either side of the lesion and the UvrD helicase removes the oligonucleotide, allowing the DNA Polymerase I to "re-synthesize" a new DNA using the complementary strand of DNA as template. [7][8][9][10][11][12][13] To complete the repair process, the LigA ligase joins the newly synthesized DNA to the adjacent pre-existing strand (Fig. 1).…”