A, Transcription initiation pathway. Core RNA polymerase (RNAP; gray oval, with active site depicted as a circle) binds to a promoter specificity σ initiation factor (1 of many) that directs the resulting holo RNAP to a subset of promoters. In Escherichia coli, the primary σ⁷⁰ factor consists of separate domains (numbered 1– 4); domains 2 and 4 recognize the −10 and −35 elements of “housekeeping” promoters to form a closed promoter complex, RP_c, in which the double-stranded DNA is loosely bound on the surface of RNAP. In RP_I1, the first intermediate along the pathway, DNA strand separation initiates around the −11 position (relative to the transcription start site). Melting propagates toward the active site (as shown in RP_I2, but additional complexes also may exist). In the final, transcription-competent open promoter complex (RP_o; boxed), the transcription bubble encompasses the +1 position. B, Order-of-addition experiments. Fidaxomicin (FDX; at 50 µM) was added to the in vitro transcription reaction with the E. coli RNAP at different points. The fraction of synthesized RNA was measured (as percentage of transcription in the absence of the antibiotic). Inhibition of the reaction was observed when FDX was added before steps 1 and 2, but not 3 or 4, after formation of the RP_o. Abbreviations: [α³²P]GTP, α-radiolabeled GTP; ApU, Adenylyl (3′-5′) uridine; ApUpC³²pG, tetranucleotide reaction product; CTP, cytidine triphosphate; FDX, fidaxomicin; GTP, guanidine triphosphate; RP_o^★, open complex stabilized by the addition of a dinucleotide primer ApU.

Fidaxomicin (FDX) inhibits DNA strand separation in promoter complexes. Melting of the promoter DNA was probed by single-hit permanganate footprinting. Potassium permanganate (KMnO₄) modifies single-stranded thymidine (T) residues, which subsequently are cleaved by piperidine; positions of cleavage indicate the location of single-stranded DNA regions. A linear end-labeled λP_R promoter DNA fragment was generated by polymerase chain reaction. Top, The −35 and −10 hexamers are indicated by black boxes, the start site (+1) is shown by a black dot. Reactivities of the −10, −4, −3, and +2 non-template strand T residues to KMnO₄ modification are summarized above the sequence; black and white arrows indicate high and low reactivity, respectively. Bottom, A representative gel with the sensitive residues shown; the middle portion of the gel was removed (dashed line) to conserve space. Results show that although myxopyronin blocks only DNA melting near the start site (at +2 position), FDX reduces strand separation throughout the entire sensitive region with the wild-type RNAP, but not with a mutant that carries an R337A substitution in the β′ switch-2 region. Abbreviations: Ab, antibiotic; FDX, fidaxomicin; MYX, myxopyronin; RNAP, RNA polymerase; WT, wild-type.