Structures and mechanism of transcription initiation by bacterial ECF factors

Abstract

Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE-the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.

Figure 1.

The cryo-EM structure of Escherichia coli σ^E-RPo. (A) The nucleic-acid scaffold used for structure determination. (B) Schematic diagram of E. coli σ^E. (C) The side and top views of cryo-EM electron density map (gray surface) at resolution 4.0 Å and the structure model of σ^E-RPo. (D) The side and top views of the overall structure of E. coli σ^E-RPo. (E) The electron density map and structural model of E. coli σ^E. (F) The electron density map and structural model of the nucleic-acid scaffold. RNAP-α subunit, light brown; RNAP-β subunit, gray; RNAP-β’ subunit, dark gray; RNAP-ω subunit, pink; σ^E₂, light green; the σ_3.2-like linker of σ^E (σ^E_3.2), cyan; σ^E₄, green. Template DNA, red; non-template DNA, orange; RNA, blue; the −35 element DNA, light blue; the −10 element, violet.

Figure 2.

The interactions among RNAP core enzyme, σ^E and nucleic-acid scaffold. (A) The βFTH interacts with a large hydrophobic surface created by σ^E₄ and the σ_3.2-like linker of σ^E. (B) The structural comparison of interactions between βFTH and domain σ₄ of Escherichia coli σ^E-RPo (green and dark gray), E. coli σ^A-RNAP (PDB ID: 6CA0; yellow) and Mycobacterium tuberculosis σ^H-RPo (PDB ID: 5ZX2; gray). (C) The E. coli σ^E–RPo (green) is superimposable to the crystal structure of E. coli σ^E₄/-35 dsDNA (gray). (D) The E. coli σ^E-RPo (yellow) is superimposable to the crystal structure of E. coli σ^E₂/-10 ssDNA (gray); (E) The detailed interaction between the E. coli σ^E₂ and the −10 element promoter DNA. (F) The stopped-flow experiments measuring the kinetics of promoter unwinding by WT or derivatives of E. coli σ^E-RNAP. The data points were recorded every 0.1 s and the data were fitted as described in ‘Materials and Methods’ section. The experiments were repeated for three times and representative curves are shown. (G) The σ_3.2-like linker of σ^E (σ^E_3.2) inserts into the active-center cleft. (H) The detailed interactions between σ^E_3.2 and template ssDNA of the transcription bubble.

Figure 3.

The crystal structure of Mycobacterium tuberculosis σ^H-RPo and σ^H/E-RPo. (A) The nucleic-acid scaffold used for the structure determination. (B) The schematic diagram of M. tuberculosis σ^H and interaction of the σ_3.2-like linker of σ^H (σ^H_3.2) with RNAP active-center cleft. (C) The schematic diagram of M. tuberculosis σ^H/E and interaction of σ^E_3.2 with RNAP active-center cleft. (D) The domain σ_3.2 of Ec σ^E, Ec σ⁷⁰, Mtb σ^E, Mtb σ^L, Mtb σ^H factors follow similar path to enter and exit RNAP active-center cleft.

Figure 4.

The sequence alignment and secondary-structure prediction of 27,670 bacterial ECF σ factors. (A) The sketched diagram of the MSA of 27,670 bacterial ECF σ factors. Upper panel, the schematic diagram of Escherichia coli σ^E; middle panel, the conservation score from the MSA for each position of E. coli σ^E; bottom panel, the occupancy score from MSA for each position of E. coli σ^E. The conservation and occupancy scores were calculated by Jalview. The occupancy scores show the ratio of ungapped positions in each column of the alignment. (B) The structures of the head, middle and tail sub-regions of Ec σ^E_3.2, Mtb σ^E_3.2, Mtb σ^H_3.2 (PDB ID: 5ZX3) and Mtb σ^L_3.2 (PDB ID: 6DV9), and Ec σ⁷⁰_3.2(PDB ID: 6CA0). (C) The primary protein sequences and secondary structures of E. coli σ^E_3.2 and Mtb σ^E_3.2. (D) The probability score of secondary structures of σ_3.2-like linkers of bacterial ECF σ factors at corresponding positions of σ^E_3.2.

Figure 5.

The σ_3.2-like linker plays essential roles in multiple steps of transcription initiation. (A) The in vitro transcription assay showing the transcription activity of WT and derivatives of various bacterial ECF σ factors. RO represents the run-off transcripts. (B) The FP competitive assay showing binding affinities of WT and derivatives of Escherichia coli σ^E to bacterial RNAP core enzyme. The experiments were repeated in triplicate, and the data are presented as mean ± S.E.M. (C) The stopped-flow experiments measuring the kinetics of promoter unwinding by WT or derivatives of E. coli σ^E. The data points were recorded every 0.1 s and the data were fitted as described in ‘Materials and Methods’ section. The Ec σ^E head region (residues 88–98) was replaced by ‘GGSSGSGGSSS’ resulting in Ec σ^E (head); the σ^E_3.2 tail region (residues 119–130) was replaced by ‘GGSSGSGGGSSS’ resulting in Ec σ^E (tail); E σ^E_3.2 head region (residues 88–98) and tail region (residues 119–130) were replaced by ‘GGSSGSGGSSS’ and ‘GGSSGSGGGSSS’, respectively resulting in Ec σ^E (head/tail). (D) The in vitro transcription assay with WT or derivatives of E. coli σ^E. The ‘abortive’ represents abortive transcripts and the ‘T’ represents terminated transcripts of 82 nt. The in vitro transcription and stopped-flow experiments were repeated for three times and representative data are shown. The FP competitive experiments were repeated for three times and the data were presented as mean ± S.E.M.