Structural basis of RfaH-mediated transcription-translation coupling

Abstract

The NusG paralog RfaH mediates bacterial transcription-translation coupling in genes that contain a DNA sequence element, termed an ops site, required for pausing RNA polymerase (RNAP) and for loading RfaH onto the paused RNAP. Here, we report cryo-electron microscopy structures of transcription-translation complexes (TTCs) containing Escherichia coli RfaH. The results show that RfaH bridges RNAP and the ribosome, with the RfaH N-terminal domain interacting with RNAP and the RfaH C-terminal domain interacting with the ribosome. The results show that the distribution of translational and orientational positions of RNAP relative to the ribosome in RfaH-coupled TTCs is more restricted than in NusG-coupled TTCs because of the more restricted flexibility of the RfaH interdomain linker. The results further suggest that the structural organization of RfaH-coupled TTCs in the 'loading state', in which RNAP and RfaH are located at the ops site during formation of the TTC, is the same as the structural organization of RfaH-coupled TTCs in the 'loaded state', in which RNAP and RfaH are located at positions downstream of the ops site during function of the TTC. The results define the structural organization of RfaH-containing TTCs and set the stage for analysis of functions of RfaH during translation initiation and transcription-translation coupling.

Fig. 1.. Structures of NusG-containing TTCs and nucleic-acid scaffolds for structure determination of RfaH-containing TTCs

(a) NusG-containing TTCs. Left: In NusG-containing collided TTC, NusG makes no interactions with ribosome (NusG-TTC-A; PDB 6VU3)^–. Center: In NusG-coupled TTC, NusG bridges TEC and ribosome (NusG-TTC-B; PDB 6XII)^–. Right: In NusA-, NusG-coupled TTC, NusG bridges TEC and ribosome, and NusA forms second bridge between TEC and ribosome. (NusA-NusG-TTC-B; PDB 6X6T). Images show EM density (gray surface) and fit (ribbons) for TEC, NusG, and NusA (at top; direction of transcription indicated by arrow in the left panel and directly toward viewer in center and right panels) and for ribosome 30S and 50S subunits and P- and E-site tRNAs (at bottom). RNAP β’, β, α^I, α^II, and ω subunits are in pink, cyan, light green, green, and gray; 30S subunit, 50S subunit, P-site tRNA, and E-site tRNA are in yellow, gray, green, and orange; DNA nontemplate strand, DNA template strand, and mRNA are in black, blue, and brick-red. NusG, NusA, and ribosomal protein S10 are in red, light blue, and magenta. Rbosome L7/L12 stalk omitted for clarity in this and subsequent images. (b) Nucleic-acid scaffolds for structure determination of RfaH-containing collided TTC (RfaH-TTC-A) and RfaH-coupled TTCs on ops-site-containing DNA (RfaH-TTC-B^ops). Each scaffold comprises nontemplate-strand oligodeoxyribonucleotide (black, with ops-site nucleotides in gray and underlined), template-strand oligodeoxyribonucleotide (blue), and one of four oligoribonucleotides having spacer lengths, n, of 7, 8, 9, and 10 codons, corresponding to mRNA (brick red). The different spacer lengths enable direct assessment of effects of differences in RNAP-ribosome spacing on transcription-translation coupling. Dashed black box labeled “TEC,” portion of nucleic-acid scaffold that forms TEC upon addition of RNAP (10 nt nontemplate- and template-strand ssDNA segments forming “transcription bubble,” 10 nt of mRNA engaged with template-strand DNA as RNA-DNA “hybrid,” and 5 nt of mRNA, on diagonal, in RNAP RNA-exit channel); dashed black lines labeled “ribosome P-site,” mRNA AUG codon intended to occupy ribosome active-center P site upon addition of ribosome and tRNA^fMet; “spacer,” mRNA spacer between TEC and AUG codon in ribosome active-center P site. (c) Nucleic-acid scaffolds for structure determination of RfaH-coupled TTCs on non-ops-site-containing DNA (RfaH-TTC-B^non-ops). Colors as in b.

Fig. 2.. Structure of RfaH-containing collided TTC

(a) Structure of RfaH-TTC-A (n = 7; Table S1). Two orthogonal views. RfaH, red. Other colors as in Fig. 1a. Dashed rectangle, region shown in b. (b) RNAP-ribosome interface in RfaH-TTC-A, showing RNAP β’ zinc binding domain, (ZBD, pink; Zn²⁺ ion as black sphere), RNAP β flap, cyan, RNAP β flap tip helix (β FTH; disordered residues indicated by cyan dashed line), and RNAP α^I (light green) interacting with ribosomal proteins S4 (forest green), S3 (orange), and S10 (magenta) and with mRNA (brick red). Portions of RNAP β’ and ribosome 30S not involved in interactions are shaded pink and yellow, respectively.

Fig. 3.. Structures of RfaH-containing coupled TTCs on ops-site-containing DNA

(a) Structure of RfaH-TTC-B^ops (n = 8; Table S1). Views and colors as in Fig. 2a. (b) Protein-DNA interactions between RfaH-N and ops-site DNA in RfaH-TTC-B^ops. (c) RNAP-ribosome interface and RfaH bridging in RfaH-TTC-B^ops (n = 8; identical interfaces for n = 7, 8, 9, and 10). RNAP β’ zinc binding domain (ZBD, pink; Zn²⁺ ion as black sphere) interacts with ribosomal protein S3 (orange) and mRNA (brick red). RfaH (red) bridges RNAP and ribosome, with RfaH-N interacting with RNAP and RfaH-C interacting with ribosomal protein S10 (magenta). Portions of RNAP β’, β, and ribosome 30S not involved in interactions are shaded pink, cyan, and yellow, respectively. (d) As c, showing cryo-EM density as blue mesh. Arrows, structural features that increase structural rigidity as compared to NusG-coupled TTC-B. Left arrow, interactions between RfaH-C and RNAP β’ ZBD. Right arrow, interactions between RfaH-N two-strand β-sheet and RfaH interdomain linker. (e) Structure of NusA-RfaH-TTC-B^ops (n = 8; Table S1). NusA in light blue. Other colors as in a. (f) Protein-DNA interactions between RfaH-N and ops-site DNA in NusA-RfaH-TTC-B^ops. (g) RNAP-ribosome interface, RfaH bridging, and NusA binding in NusA-RfaH-TTC-B^ops (n = 8; identical interfaces for n = 8, 9, and 10). RfaH (red) bridges RNAP and ribosome, with RfaH-N interacting with RNAP and RfaH-C interacting with ribosomal protein S10 (magenta). NusA (light blue) KH1 domain interacts with ribosomal proteins S5 and S2 (brown and forest green). Portions of RNAP β’, β, and ribosome 30S not involved in interactions are shaded pink, cyan, and yellow, respectively. (h) As g, showing cryo-EM density as blue mesh.

Fig. 4.. Structures of RfaH-containing coupled TTCs on non-ops-site-containing DNA

(a) Structure of RfaH-TTC-B^non-ops (n = 8; Table S1). Views and colors as in Fig. 2a and 3a. (b) Interactions between RfaH-N and non-ops-site DNA in RfaH-TTC-B^non-ops. (c) RNAP-ribosome interface and RfaH bridging in RfaH-TTC-B^non-ops. RNAP β’ zinc binding domain, (ZBD, pink; Zn²⁺ ion as black sphere) interacts with ribosomal protein S3 (orange) and mRNA (brick red). RfaH (red) bridges RNAP and ribosome, with RfaH-N interacting with RNAP and RfaH-C interacting with ribosomal protein S10 (magenta). Portions of RNAP β’, β, and ribosome 30S not involved in interactions are shaded pink, cyan, and yellow, respectively. (d) As c, showing cryo-EM density as blue mesh. Arrows, structural features that increase structural rigidity as compared to NusG-coupled TTC-B. Left arrow, interactions between RfaH-C and RNAP β’ ZBD. Right arrow, interactions between RfaH-N two-strand β-sheet and RfaH interdomain linker. (e) Structure of NusA-RfaH-TTC-B^{non-ops-containing} (n = 8; Table S1). NusA in light blue. Other colors as in a. (f) Interactions between RfaH-N and non-ops-site DNA in NusA-RfaH-TTC-B^non-ops. (g) RNAP-ribosome interface, RfaH bridging, and NusA binding in NusA-RfaH-TTC-B^non-ops. RfaH (red) bridges RNAP and ribosome, with RfaH-N interacting with RNAP and RfaH-C interacting with ribosomal protein S10 (magenta). NusA (light blue) KH1 domain interacts with ribosomal proteins S5 and S2 (brown and forest green). Portions of RNAP β’, β, and ribosome 30S not involved in interactions are shaded pink, cyan, and yellow, respectively. (h) As g, showing cryo-EM density as blue mesh.