Canonical bacterial transcription activators bind to their cognate cis elements at the upstream of transcription start site (TSS) in a form of dimer. Caulobacter crescentus GcrA, a non-canonical transcription activator, can activate transcription from promoters harboring its cis element at the upstream or downstream of TSS in a form of monomer. We determined two cryo-EM structures of C. crescentus GcrA-bound transcription activation complexes, GcrA TAC(U) and GcrA TAC(D), which comprise GcrA, RNAP, sigma(70) and promoter DNA with GcrA cis elements at either the upstream or downstream of TSS at 3.6 and 3.8 angstrom, respectively. In the GcrA-TAC(U) structure, GcrA makes bipartite interactions with both sigma(70) domain 2 (sigma(70)(2)) and its cis element, while in the GcrA-TAC(D) structure, GcrA retains interaction with sigma(70)(2) but loses the interaction with its cis element. Our results suggest that GcrA likely forms a functionally specialized GcrA-RNAP-sigma(A) holoenzyme, in which GcrA first locates its cis element and then facilitates RNAP to load on core promoter at its proximal region. The sequence-specific interaction of GcrA and DNA is disrupted either at the stage of RPo formation or promoter escape depending on the location of GcrA cis elements relative to TSS.
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