Catching RNA polymerase in the act of binding: Intermediates in transcription illuminated by synchrotron footprinting

T he article by Sclavi et al. (1) in this issue of PNAS addresses “initiation,” the first step in transcription. Gene transcription is catalyzed in cells by large multisubunit proteins called RNA polymerases (RNAP). The eubacteria holoenzyme of RNAP is composed of five core subunits (α, α2, β, β′, and ω) that contain the amino acid residues required for the enzyme's catalytic activity. A sixth subunit (σ) guides RNAP to specific sequences on the genomic DNA (promoters) that mark the beginning of a gene or group of genes. Transcription consists of three phases: initiation, elongation, and termination, with each of these phases being highly regulated by DNA/RNA sequence and/or accessory proteins (Fig. 1). RNAP holoenzyme must first locate a promoter. After binding to the promoter, the RNAP holoenzyme and the bound DNA undergo a series of conformational changes from the closed to the open promoter complex, in which ≈12 base pairs of DNA denature to form a bubble of the two separated strands of DNA. Fig. 1. A schematic outline of the transcription cycle phases at the operon level of genomic organization. RNAP holoenzyme binds to the promoter and initiates RNA synthesis. The σ-subunit is released at the onset of elongation. RNA synthesis continues without dissociation of the RNAP core until the terminator sequence is reached when the RNA transcript and polymerase are released. The expanded diagram beneath the schematic depicts RNAP holoenzyme binding to a promoter and its isomerization to an open complex. RNAP holoenzyme is now poised to initiate RNA synthesis. After the addition of 6-10 RNA nucleotides, the σ-subunit is released, and the RNAP core continues to elongate the RNA chain until reaching a DNA terminator sequence. The terminator induces dissociation of the RNAP core and the completed RNA transcript from the DNA. RNAP core reassociates with …