Remarkably versatile chemistry of Bodipy dyes allows the design and straightforward synthesis of multivalent-multitopic derivatives, which, with judicious selection of metal ion-ligand pairs based on known affinities, affords control and manipulation of photoinduced electron transfer and internal charge transfer processes as desired. We have demonstrated that metal ions acting as modulators (or inputs, in digital design parlance) can generate absorbance changes in accordance with the operation of a halfadder. In addition, an AND logic gate in the emission mode was delivered using a different binucleating arrangement of ligands. A molecular equivalent of a three-input AND logic gate was also obtained exploiting differential binding affinities of metal ions for different ligands. The results suggest that different metal ions can be used as nonannihilating inputs, selectively targeting various ligands incorporated within a single fluorophore, and with careful design, diverse photophysical processes can be selectively modulated, resulting in a range of signals, useful in molecular logic design, and offering an enticing potential for multianalyte chemosensors.

A key regulated step of transcription in all cellular organisms is promoter melting by the RNA polymerase (RNAP) to form the open promoter complex1–3. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12–14 base pairs (bps) of the DNA duplex, and load the template-strand (t-strand) DNA into the RNAP active site. Formation of the open complex is a multi-step process with transient intermediates of unknown structures4–6. Here, we present cryo-electron microscopy structures of bacterial RNAP-promoter DNA complexes, including the first structures of partially melted intermediates. The structures unequivocally show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 (FL2) and switch 2 (Sw2), universal structural features of RNAP, in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of the FL2 and Sw2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression in all domains of life.

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against Mycobacterium tuberculosis RNAP in vitro, but clinical use of Fdx is limited to treating Clostridium difficile intestinal infections due to poor absorption. To identify the structural determinants of Fdx binding to RNAP, we determined the 3.4 Å cryo-electron microscopy structure of a complete M. tuberculosis RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin, explaining its strong effect on M. tuberculosis. Additional structures define conformational states of M. tuberculosis RNAP between the free apo-holoenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on the Fdx binding pocket.

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