Diverse and unified mechanisms of transcription initiation in bacteria

Abstract

Transcription of DNA is a fundamental process in all cellular organisms. The enzyme responsible for transcription, RNA polymerase, is conserved in general architecture and catalytic function across the three domains of life. Diverse mechanisms are used among and within the different branches to regulate transcription initiation. Mechanistic studies of transcription initiation in bacteria are especially amenable because the promoter recognition and melting steps are much less complicated than in eukaryotes or archaea. Also, bacteria have critical roles in human health as pathogens and commensals, and the bacterial RNA polymerase is a proven target for antibiotics. Recent biophysical studies of RNA polymerases and their inhibition, as well as transcription initiation and transcription factors, have detailed the mechanisms of transcription initiation in phylogenetically diverse bacteria, inspiring this Review to examine unifying and diverse themes in this process.

Figure 1.. Overview of the steps of bacterial transcription initiation and RNAP.

(A) Schematic of bacterial transcription initiation. The cartoon shows steps of bacterial transcription initiation, starting from core RNAP to the elongation complex. The core RNAP (colored in grey with the active site Mg²⁺ depicted as a green dot) binds a promoter specificity factor, σ (orange), generating the holoenzyme. Holoenzyme recognizes and melts promoter DNA (blue) to form the open promoter complex. In the presence of rNTP, the initiation complex scrunches and can synthesize many RNA transcripts (red) in a process called abortive transcription. Eventually, the complex produces a transcript length that competes with σ, which in combination with scrunching, causes σ to disassociate from the core RNAP allowing it to escape the promoter. The enzyme then transitions to the elongation complex and completes transcription of the gene. (B) Architecture the active site Mg²⁺, the main channel, the secondary channel, the bridge helix (in cartoon tubes), the β lobe, the β protrusion, and the β’ clamp highlighted. (C) Holoenzyme configuration and recognition of housekeeping promoter. (Left) Holoenzyme is shown as a transparent molecular surface and colored according to the color key with σ, the β flap-tip helix, and the clamp helices shown as cartoon tubes. The regions of holoenzyme that bind the promoter elements are highlighted: αCTDs [pale green] bind the UP element [teal], σD4 binds −35 element [yellow], σD3.2 is buried in the RNAP active site cleft, σD3 interacts with the extended −10 [EXT, blue], σD2 interacts with the −10 [magenta], σNCR is located between σD2 and σD1.2, σD1.2 interacts with the discriminator region [DSR, forest green], and the β subunit of RNAP recognizes the core recognition element [CRE, light blue]. The TSS is shown as a ‘+1’, and the direction of transcription is depicted by the black arrow below the TSS. (Right) Structure of open promoter complex primed for initiation showing the promoter DNA placed into RNAP with an RNA primer.

Figure 2.. Structure-based model of RPo formation highlighting RNAP motions that lead to promoter melting.

Structural models of RNAP were generated from cryo-EM structures of promoter melting intermediates from E. coli (PDB IDs 6PSQ, 6PSS, 6PST, 6OUL) and M. tuberculosis (PDB ID 6EE8) with lineage-specific inserts removed for clarity. Proteins are shown as transparent molecular surface, while σD1.1 and promoter DNA are shown as spheres. The Mg²⁺ (shown as a green sphere) marks the active site. The pathway is composed of structures delineating bubble propagation that leads to the open promoter complex.

Figure 3.. Factors that regulate the pool of holoenzymes.

(A) Despite divergent sequence, structure, and phylogenetic origins, anti-σs (red) prevent binding of the alternative σ by occluding the major core binding interface (σD2, green) from the core β’-clamp helices (pink). The β’-clamp helices were modeled by superimposing σD2 from structures of the σ/anti-σ complexes unto the σ^A holoenzyme structure (PDB ID 19LU). Structures shown and numbered are listed with PDB IDs in parentheses as follows: (1) Rhodobacter sphaeroides σ^E/ChrR (PDB ID 2Q1Z), (2) E. coli σ^E/RseA (PDB ID 1OR7), (3) P. aeruginosa σ^H/MucA (PDB ID 6IN7), (4) B. subtilis σ^W/RsiW (PDB ID 5WUR), (5) M. tuberculosis σ^K/RskA (PDB ID 4NQW), (6) B. Quintana σ^E/NepR (PDB ID 5UXX), (7) Caulobacter vibroides σ mimic PhyR/NepR (PDB ID 3T0Y), (8) Cupriaviuds metalliiduruns σ^CnrH/CnrY (PDB IDm4CXF), (9) Sf. venezuelae σ^BldN/RsbN (PDB ID 6DXO), (10) S. venezuelae σ^WhiG/RsiG (PDB ID 6PFJ) and (11) Aquifex aeolicus σ^FliA/FlgM (PDB ID 1SC5). (B) Schematics of the three founding members of σ-tethers (purple), tethering σD2 and σNCR to the core enzyme and or promoter DNA. (C) 6S RNA mimics the promoter architecture of an open complex, sequestering the housekeeping σ holoenzyme. Shown are the cryo-EM structures of 6S/holoenzyme (PDB ID 5VT0) and the crystal structure σ holoenzyme with promoter DNA (PDB ID 4YLN).

Figure 4.. Diversity in bacterial RNAPs and their regulation.

(A) Structures of T. aquaticus RNAP (PDB ID 4XLR), E. coli RNAP (PDB ID 4LK1), and M. tuberculosis RNAP (PDB ID 6C05); highlighting the disposition of lineage-specific inserts. RNAPs are shown as molecular surfaces. Lineage-specific inserts are shown as transparent molecular surfaces superimposed with cartoon tubes (T. aquaticus lineage-specific inserts, green; E. coli lineage-specific inserts, blue; M. tuberculosis lineage-specific inserts, magenta). (B) Examples of unconventional transcription factors that regulate transcription initiation. Proteins are shown as a transparent molecular surface. Promoter DNA is shown as cartoons superimposed on a transparent molecular surface. The active site Mg²⁺ is shown as a green sphere. (Left) E. coli RNAP bound to promoter DNA and TraR (PDB ID 6PSV). TraR (red) is shown as cartoon tubes. Highlighted is the interaction between TraR and the Escherichia coli RNAP lineage-specific inserts: βi4 and β’i6 (colored blue). (Right) M. tuberculosis open promoter complex (PDB ID 6EDT) bound to RbpA (purple) and CarD (green). Highlighted are the CarD W85 that “wedges” the upstream fork junction and the RbpA N-terminal tail (NTT) that interacts with the T-strand that is loaded in the RNAP active site. β’i1 is also shown and colored magenta.

Figure 5.. Inhibitors of RNAP.

A) The positions of the antibiotics that bind RNAP. Sorangicin and kanglemycins are not shown as they overlap with rifampicin. The bridge helix (BH) and trigger loop (TL) are drawn in cartoon as indicated. The active site Mg²⁺ is shown for reference. B) The minimal mechanism for transcription initiation is shown, and the steps inhibited by each antibiotic is indicated.