The bacterial enhancer-dependent RNA polymerase

Abstract

Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript.

Keywords: AAA+ proteins; RNA polymerase; bEBPs; sigma54; sigma70; transcription.

Figure 1.. Structure of σ⁵⁴-RNAP.

(A) Domain organization of σ⁵⁴. NMR structures of the CBD and RpoN domains are illustrated (PDB entries 2K9L and 2O8K). (B) Crystal structure of σ⁵⁴ (PDB entry 5BYH). Individual domains are coloured as in (A). (C–E) σ⁵⁴-RNAP holoenzyme in different orientations, coloured by subunits, α — grey; β — wheat; β′ — teal; ω — light pink. The dotted line in (E) depicts the trajectory of the σ⁵⁴ RpoN domain in (D) before binding to the promoter.

Figure 2.. Unique functional domains and their interaction partners.

(A) The σ⁵⁴ CBD domain interacts with α-CTD, β-flap, β-C-terminus (Cter), β′-dock, and β′ zipper. (B) The σ⁵⁴ RpoN domain does not seem to interact with other parts of RNAP. (C) σ⁵⁴ RI and RIII ELH/HTH lie directly above the cleft formed between β and β′. (D) Fitting of σ⁵⁴-RNAP crystal structure into σ⁵⁴-RNAP-bEBPs cryo-EM contour. RI (and possibly RIII) lies at the connecting density leading to hexameric bEBPs. (E) σ⁵⁴ RII is buried deeply in the DNA-binding channel and directly above the bridge helix and trigger loop. (F and G) σ⁵⁴ RII.2 and 3 (boxed) coincide with the path of the DNA template strand. RII.2 may need to be relocated for RNA extension to occur. The PDB entry 4YLN was used to model the DNA/RNA path in (G).

Figure 3.. Structural comparison of three RNAPs.

Comparison of σ⁵⁴-RNAP (PDB entry 5BYH, A), σ⁷⁰-RNAP (PDB entry 4IGC, B), and Pol II–TFIIB (PDB entry 4BBS, C). ZnRib stands for Zn ribbon domain.

Figure 4.. Interactions with the β′-coiled-coil domain.

σ⁷⁰ 2–3 (A) and TFIIB linker core (B) form helical bundles with the β′-coiled-coil domain. In contrast, σ⁵⁴ RI–RIII (C) does not form a helical bundle with the β′-coiled-coil domain. (D) The β′ i6 domain is visible in a σ⁵⁴-RNAP crystal structure. It sandwiches the β′-jaw domain with the β′-clamp.

Figure 5.. Comparison of σ⁵⁴-RNAP, σ⁷⁰-RNAP, Pol II–TFIIB, and Pol I (PDB entry 4C2M).

(A–C) σ⁵⁴ RII3, σ⁷⁰ 3.2, and TFIIB linker all occupy similar space within the holoenzyme. (D) Sequence alignment of σ⁵⁴ RII from Klebsiella pneumonia (Kp), E. coli (Ec), Pseudomonas putida (Pp), Rhodobacter capsulatus (Rc), Salmonella typhimurium (St), A. aeolicus (Aa), and σ⁷⁰ 3.2 from Ec. The conserved DDE motif is boxed. The DNA passage into the DNA-binding channel is blocked by σ⁵⁴ RI–RIII (E), but allowed by σ⁷⁰ 2–3 ‘v’ gate (F) and TFIIB linker core (G). (H) The A14-43 dimer from an adjacent molecule occupies a similar space as does σ⁵⁴ RI–RIII (compare with E). The expander sequence occupies a similar space as σ⁵⁴ RII.2/3.

Figure 6.. Transcription regulation hotspots in RNAP and regulations by sigma factors, TFIIB, and those in Pol I.

Those shared by others are in brackets (blue — sigma70; green — Pol I; red — Pol I–II–III–TFIIB).