Fig 1.
Cryo-EM reconstructions of the class-II CAP-TAC without RNA transcript.
(A) Schematic representation of the synthetic promoter DNA scaffold (78 bp) in the class-II CAP-TAC. (B-C) Overviews of the cryo-EM reconstruction maps of the E. coli class-II CAP-TAC without RNA transcript at 4.5 Å (B, state 1) and 4.3 Å (C, state 2) resolutions, respectively. The individually colored density maps, created by color zone, split in Chimera, and shown in a contour of 8 root-mean-square (RMS), are displayed in transparent surface representation to allow visualization of all the components of the complex. CAP-TAC, CAP-dependent transcription activation complex; cryo-EM, cryo–electron microscopy; NCR, non-conserved region; NT, non-template; αCTD, carboxyl-terminal domain of the alpha subunit; αNTD, amino-terminal domain of the alpha subunit.
Fig 2.
Structural comparisons reveal conformational changes in RNAP.
(A) Superimposition of the state 1 CAP-TAC with the previous RPo (PDB 6CA0) [21] via the σ2 and σ3 domains of σ70. The components are shown in pipes and planks representation. The state 1 and RPo structures are shown in orange and cyan, respectively, except for dark gray (state 1) and light gray (RPo) DNAs. (B) The close-up views of the main cleft along two directions. The DNA promoter from RPo and all the σ70 proteins are omitted for clear representation. The movement directions and maximal distances of the secondary structures in the domains surrounding the main cleft are labeled using magenta arrows and specific values, respectively. (C) Superimposition between the state 1 (orange and dark gray) and state 2 (cyan and light gray) CAP-TAC without RNA transcript via the σ2 and σ3 domains of σ70. CAP-TAC, CAP-dependent transcription activation complex; NCR, non-conserved region; NTP, nucleoside triphosphate; PDB, Protein Data Bank; RNAP, RNA polymerase; RPo, RNAP-promoter open complex; SI1, sequence insertion 1; αNTD, amino-terminal domain of the alpha subunit.
Fig 3.
Cryo-EM reconstruction of the class-II CAP-TAC with de novo RNA transcript.
(A) Overview of the cryo-EM reconstruction map of the E. coli class-II CAP-TAC with RNA transcript at 4.4 Å resolution and the state 2 architecture. The color schemes for the split density maps (8 RMS) and the docked components are same as in Fig 1. (B) A close-up view of the promoter DNA scaffold in the complex. The insert is the zoom-in view of the DNA-RNA hybrid region with the magenta Mg2+ sphere. A de novo synthesized RNA transcript (3-nucleotide) starting from the −1 position with a GTP residue is displayed. CAP-TAC, CAP-dependent transcription activation complex; cryo-EM, cryo–electron microscopy; NCR, non-conserved region; RMS, root-mean-square.
Fig 4.
Hypothesized mechanism of transcription activation on class-II promoters.
A schematic cartoon model of CAP activating transcription on class-II DNA promoters is presented. When the CAP dimer interacts with RNAP holoenzyme and the class-II DNA promoter, it may either induce conformational changes in RNAP and consequently significantly widen the main cleft to form a state 1 architecture or stabilize the naturally occurred intermediate, which might facilitate the DNA promoter entering into the main cleft. The complex at the state 1 can convert to the one with the state 2 architecture that contains narrow main cleft during the formation of the RPo. With transcription initiation and the synthesis of RNA transcript, all the complexes with different states would convert to the ones with the state 2 architecture. The colored arrows in the rectangle indicate the individual movement directions. CAP, cAMP receptor protein; NTP, nucleoside triphosphate; RNAP, RNA polymerase; RPinit, RNAP-initiation complex; RPo, RNAP-promoter open complex.