Architecture of a transcribing-translating expressome

Abstract

DNA transcription is functionally coupled to messenger RNA (mRNA) translation in bacteria, but how this is achieved remains unclear. Here we show that RNA polymerase (RNAP) and the ribosome of Escherichia coli can form a defined transcribing and translating "expressome" complex. The cryo-electron microscopic structure of the expressome reveals continuous protection of ~30 nucleotides of mRNA extending from the RNAP active center to the ribosome decoding center. The RNAP-ribosome interface includes the RNAP subunit α carboxyl-terminal domain, which is required for RNAP-ribosome interaction in vitro and for pronounced cell growth defects upon translation inhibition in vivo, consistent with its function in transcription-translation coupling. The expressome structure can only form during transcription elongation and explains how translation can prevent transcriptional pausing, backtracking, and termination.

Fig. 1. Reconstitution of the expressome

Template DNA, non-template DNA and mRNA carrying a ribosome-binding sequence (RBS) and a 3′-desoxy-C (dC) end were annealed to obtain a nucleic acid scaffold. The mRNA register is indicated in red, where +1 is the nucleotide addition site in RNAP and negative numbers indicate upstream positions with respect to this site. RNAP was added to form a stalled transcription elongation complex (EC). The EC was then used as a template for in vitro translation. The ribosome translated the mRNA until encountering the stalled RNAP. Expressomes were purified from the reaction mix.

Fig. 2. Architecture of the expressome

(A) Cryo-EM reconstruction of the expressome. The electron density map was low-pass filtered to 9 Å resolution. Structures were rigid-body fitted. Ribosome landmarks are labeled in black (L1 St, L1 stalk; h, head; pt, platform). Color codes: 50S ribosomal subunit (blue), 30S (yellow). 30S proteins rS2 (pink), rS4 (dark blue), rS5 (forest green), rS9 (cyan), RNAP subunit α1 (light grey), α2 (dark grey), β (light blue), β′ (salmon), ω (light purple), template DNA (dark blue), non-template DNA (cyan), mRNA (red), codon and anticodon nucleotides (light rose), P-site tRNA (dark green), nascent peptide (orange). (B) Section through the fitted structures (PDB codes 2avy, 2aw4, and 5byh without αCTD) showing the path of mRNA (red) from the RNAP active center to the decoding center of the ribosome. The path of the nascent peptide chain is shown as orange dots (compare Fig. S). Same view as in (A). (C) Segmented density for template DNA, mRNA and tRNA. Nascent mRNA reaches the surface of the RNAP at ~register −12 and enters the ribosome at ~register −18. The mRNA codon nucleotides are at ~register −27 to −29.

Fig. 3. RNAP-ribosome interactions

(A) Footprint of the RNAP on the solvent exposed surface of the 30S ribosomal subunit. View tilted 90° relative to Fig. 2A. Ribosomal proteins (rS2, rS3, rS4, rS5, rS9, rS10, rS14) are in different colors and labeled. The RNAP footprint on the 30S is outlined in grey and subdivided in regions contacting RNAP subunits. The dashed grey oval marks a putative αCTD contact site. The red star marks the entrance to the ribosomal RNA tunnel. (B) View of the RNAP-30S interface reveals an additional density attributed to the αCTDs (dashed oval). The αCTDs apparently contact 16S rRNA near h38 (~nt 1120) and h39 (~nt 1145) and rS9. The RNAP β-SI2 helix bundle (lower left) contacts H33 of the 16S rRNA. It is apparently mobile since the observed density does not fully cover the SI2 motif of the 5byh crystal structure. H33 moves towards RNAP relative to its location in the ribosome structure (PDB code 2avy, arrow). Relative to Fig. 2A, the view is rotated counterclockwise by 110° around the vertical axis.

Fig. 4. RNAP-ribosome interaction is direct and functional

(A) RNAP variants were pre-incubated with 70S ribosomes at 1 μM concentration. Co-migration in a sucrose gradient was monitored by SDS-PAGE of ribosome-containing fractions (lanes 1–3). RNAP wt and RNAP ΔSI2 bind strongly to ribosomes, RNAP ΔαCTD shows strongly reduced binding, identifying the RNAP αCTD as an important binding module. To the right of the marker (lane 4), loading controls show the expected molecular weight of the proteins (lanes 5–8). (B) RNAP (wild-type or lacking the αCTD) was expressed from plasmids under control of the P_bad promoter in minimal medium. After 40 minutes of expression, chloramphenicol (Cm), bicyclomycin (Bcm), or both was added to inhibit ribosomes and ρ termination factor, respectively. (C) Cell density with time for cultures with or without Cm at 4 μg/ml. The reduction in the effect of Cm became more pronounced upon longer incubation.