An α helix to β barrel domain switch transforms the transcription factor RfaH into a translation factor

Abstract

NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a β barrel in NusG and an α hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here, we show that the released RfaH-CTD completely refolds from an all-α to an all-β conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated. PAPERFLICK:

Figure 1. Structures of NusG and RfaH

(A) Ribbon representation of NusG (NTD, light green, PDB-ID: 2K06; CTD, green, PDB-ID: 2JVV (Mooney et al., 2009b)). (B) Ribbon representation of full-length RfaH (NTD, light blue; CTD, blue; PDB-ID: 2OUG (Belogurov et al., 2007)), with RfaH-CTD in an α-helical fold. Termini and linker regions (broken lines) are labeled.

Figure 2. Structure of full-length RfaH in solution

(A) Ribbon representation of the crystal structure; PDB-ID: 2OUG (Belogurov et al., 2007). NTD, light blue; CTD, blue. Termini and secondary structure elements are labeled. (B) Chemical shift index (CSI) for Cα and CO vs. sequence position. Secondary structure elements as in (A). (C) R₁ for full-length RfaH. (D) R₂ for full-length RfaH. (E) R₁/R₂ is unimodal for full-length RfaH. RfaH-CTD (124-158), blue bars; RfaH-NTD (1–100), light blue bars.

Figure 3. Structural transition of RfaH-CTD

(A) Ribbon diagram of a representative RfaH-CTD structure, residues 101–162, termini and secondary structure elements are labeled. Orientation relative to Figure 1 is indicated. (B) Ensemble of 20 accepted lowest energy structures. The flexible part, residues 101 107, is indicated. (C) Ribbon representation of NusG-CTD (PDB-ID: 2JVV (Mooney et al., 2009b)). Residues in the hydrophobic core of the CTD, sticks; carbon, magenta; oxygen, red; nitrogen, blue; sulfur, yellow. (D) RfaH-CTD (PDB-ID: 2OUG (Belogurov et al., 2007)) in the all α-helical conformation; residues corresponding to the NusG-CTD core, magenta sticks. Within the α-helical RfaH-CTD these residues are scattered randomly along the helices (middle). For residues P110 and F159, no electron density could be determined in the full-length protein (Belogurov et al., 2007); therefore, these are excluded. Domain opening leads to drastic refolding of RfaH-CTD (right) into an all β-barrel state; the residues corresponding to the NusG-CTD core also form the refolded RfaH-CTD core. See also Table S1 and Figure S2.

Figure 4. Structural transition of RfaH-CTD in full-length RfaH

(A, B) Overlay of [¹H,¹⁵N]-HSQC spectra of ¹⁵N-RfaH-E48S, 45 μM, black, ¹⁵N-RfaH, 189 μM, cyan, and ¹⁵N-RfaH-CTD, 344 μM, red. Full spectra (A), and enlargement of one region (B). Signals from the CTD in full-length RfaH (α-helical form), cyan, from the isolated CTD (β-barrel form), red. (C) Overlay of [¹H,¹⁵N]-HSQC spectra of ¹⁵N-RfaH(NTD-TEV-CTD), 138 μM, black, ¹⁵N-RfaH, 189 μM, cyan, and ¹⁵N-RfaH(NTD-TEV-CTD) after incubation with 1.75 μM TEV protease for 42 hours, 127 μM, red. (D) Overlay of [¹H,¹⁵N]-HSQC spectra of ¹⁵N-RfaH(NTD-TEV-CTD) 127 μM, red. after incubation with 1.75 μM TEV protease for 42 hours from (C) and ¹⁵N-RfaH-CTD, 344 μM, black; signals of the isolated CTD in the β-barrel conformation, red.

Figure 5. RfaH-CTD is required for effects on translation and may interact with S10

(A, B) Reporter assays using the translation-competent (A, pIA955) and defective (B, pIA1087) ops-lux operon constructs, which differ in sequence 5 nt upstream from the ATG codon, GAGGA and CACAC, respectively. The assays were performed in the rfaH deletion strain transformed with plasmids encoding RfaH variants under control of the P_BAD promoter, as before (Belogurov et al., 2010). The results are expressed as luminescence corrected for the cell densities of individual cultures. (C) Expression of rfb operon (top) evaluated by qRT-PCR. Total RNA was isolated from ΔrfaH cells expressing WT or an altered RfaH variant and the absolute amount of wbbI mRNA was measured. In (A-C), errors (± standard deviation) were calculated from three independent experiments. (D–G) ChIP-chip analysis of the protein-coding rfb and atp operons and non-coding rrnE, rnpB and ssrA genes, performed as described previously (Belogurov et al., 2009; Mooney et al., 2009a) with probe sets for the rfb (D), atp (E), rrnE (F), ssrA (G), and rnpB (G), transcription units (TUs). Cy3 signal (IP) from the DNA immunoprecipitated with monoclonal antibodies to RNAP (ß-subunit), NusA, or HA-epitope tag on NusG or polyclonal antibodies to RfaH, NusE (S10), or NusB was divided by Cy5 signal (input) from unenriched DNA collected prior to immunoprecipitation. The data for each target were quantile normalized against each other so that relative signal ratios could be compared, and plotted on a log₂ scale. The ratios of average NusE/RNAP and NusG/RNAP signals were 1.1 and 0.47 (rfb), 1.1 and 0,88 (atp), 0.98 and 1.1 (rrnE), 0.35 and 0.45 (rnpB), and 0.36 and 0.55 (ssrA). See also Figure S5.

Figure 6. RfaH-CTD:S10 interface

(A) Mapping of the titration induced [¹H,¹⁵N]-HSQC chemical shift changes (Δδ_norm [ppm] >0.2, red; >0.1, orange; and >0.04, yellow) on structures of the NusB:S10 complex (dark and light gray, respectively; PDB-ID 3D3B (Luo et al., 2008)) and RfaH-CTD (gray). Strongly affected residues (sticks; carbon, red; oxygen, red; nitrogen, blue; sulfur, yellow) are shown. Gray sphere in S10 denotes the Cα position of S46, which in this construct replaces residues 46 to 67 of the WT S10 (Luo et al., 2008). (B) Surface representation of the structures shown in (A). Orientations relative to panel (A) (NusB:S10) and Figure 1 (RfaH-CTD) are indicated. Inserts show the corresponding interaction surface of the NusB:S10:NusG-CTD complex (Burmann et al., 2010). (C) [1H,¹⁵N]-HSQC-derived chemical shift changes vs. sequence position. (Left) S10 chemical shift changes on titration with RfaH-CTD; missing residues of the S10 ribosome-binding loop are indicated by a break on the sequence axis. (Right) RfaH-CTD chemical shift changes on titration with S10. Dotted line, significance level of Δδ_norm[ppm]=0.04; red bars, signals disappearing upon complex formation. Triangles, unassigned residues. See also Figure S6.

Figure 7. Model for multi-faceted activation of gene expression by RfaH

In RfaH-controlled operons, the ops element is located within 100 nt upstream of a GTG predicted (based on protein sequence analysis) to serve as a translation start codon. In absence of RfaH (left), NusG-NTD binds to the β’-clamp helices (dark gray cylinder) and NusG-CTD interacts with Rho (purple) to terminate transcription by RNAP (gray). In the rfb operon, Rho decreases expression of distal genes by ~800-fold (Sevostyanova et al., 2011). When present (right), RfaH binds to elongating RNAP at the ops site and reduces Rho effect to 2-fold. This strong anti-polar activity depends on the coordinated action of both RfaH domains becoming separated during recruitment. Unaltered RfaH-NTD binds to the β’-clamp helices to reduce transcriptional pausing and exclude NusG-NTD from binding to RNAP; both activities inhibit Rho-dependent termination. The refolded RfaH-CTD recruits the 30S subunit (bound to the initiator tRNA) via direct contacts with S10. Thus, the tethered translation initiation complex scans the mRNA lacking a strong Shine-Dalgarno (SD) element for another, yet unknown, start signal. Recruitment of the ribosome directly increases translation and indirectly decreases Rho-dependent termination by shielding mRNA from Rho.