Our data reveal two protein-protein interaction regions in CarDNt, each directed at a ABT-199 distinct protein partner. One mediates binding to RNAP-�� and corresponds to the 72-residue LY2109761 Nterminal segment, CarD1�C72, whose twisted, five-stranded ��-sheet Tudor-like fold as well as contacts with RNAP-�� closely match those observed for its CdnL counterpart. The second CarDNt module, CarD61�C179, interacts with CarG, but this protease-susceptible domain has thus far eluded a high-resolution structure determination. By contrast, the CarD61�C179 counterpart of CdnL, CdnLCt, is protease-resistant, could be expressed as a stable isolated domain, and its compact, all-helical, high-resolution tertiary structure readily determined by NMR. These differences and the inferences from far-UV CD data suggest that CarD61�C179 and CdnLCt are likely to be structurally distinct. Even so, CarD61�C179 has a stretch of basic residues that is not involved in the interaction with RNAP or CarG and yet is critical for CarD function, just as, interestingly, the equivalent conserved segment in CdnLCt is crucial for CdnL function. In sum, our data indicate that CarDNt mirrors CdnL in domain organization and in various interactions, but different contributions from these and additional interactions specific to CarD can account for its distinct function. Mutations that impair CdnL interactions with RNAP-�� were very adverse to cell growth and survival. It is therefore interesting that equivalent mutations in CarD, which mimics CdnL in RNAP-�� recognition, only slightly lowered its activity in vivo. Studies with CdnL showed that it associates with rRNA promoters in vivo and activates these by stabilizing the formation of transcriptionally competent open complexes by RNAP holenzyme with the primary housekeeping and that this activity of CdnL is impaired by mutations disrupting the interaction with RNAP-��. CdnL did not preferentially localize in vivo at PQRS, the alternative ECF-�� CarQ-dependent promoter whose activation requires CarD, suggesting absence of direct CdnL action at this promoter. By contrast, both CarD and CarDNt act on PQRS but not on rRNA promoters. The little or no effect on PQRS expression on disrupting CarD/CarDNt binding to RNAP-�� therefore suggests that this interaction, unlike with CdnL, is not as critical in transcriptional activation mediated by CarD. Other CarD interactions should then be more decisive determinants of its function. One interaction indispensable in every known CarD-dependent process is that with CarG. We mapped this interaction in the present study to the CarD61�C179 segment, but the exact molecular mechanism by which CarG acts together with CarD in ECF-�� promoter activation remains enigmatic. In the HMGA-driven assembly of the large transcriptionally competent complex in eukaryotes known as the enhanceosome, a key role is played by transcriptional factors that do not bind DNA but rather provide a protein scaffold for interaction with various other regulatory factors. CarG, which does not bind DNA directly, could collaborate with CarD in mediating the recruitment of additional factors required for promoter expression or bridge additional contacts with the basal transcriptional machinery essential for activation. We have not been able to detect direct CarG physical interactions with any of the core RNAP subunits nor with CarQ in two-hybrid analysis, but the likelihood that these occur once the CarD/CarG complex and RNAP have assembled at the target promoters cannot be discarded.