Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation

Said, Nelly; Finazzo, Mark; Hilal, Tarek; Wang, Bing; Selinger, Tim Luca; Gjorgjevikj, Daniela; Artsimovitch, Irina; Wahl, Markus C.

doi:10.1038/s41467-024-47439-6

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Published: 15 April 2024

Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation

Nature Communications volume 15, Article number: 3186 (2024) Cite this article

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Abstract

Transcription termination factor ρ is a hexameric, RNA-dependent NTPase that can adopt active closed-ring and inactive open-ring conformations. The Sm-like protein Rof, a homolog of the RNA chaperone Hfq, inhibits ρ-dependent termination in vivo but recapitulation of this activity in vitro has proven difficult and the precise mode of Rof action is presently unknown. Here, our cryo-EM structures of ρ-Rof and ρ-RNA complexes show that Rof undergoes pronounced conformational changes to bind ρ at the protomer interfaces, undercutting ρ conformational dynamics associated with ring closure and occluding extended primary RNA-binding sites that are also part of interfaces between ρ and RNA polymerase. Consistently, Rof impedes ρ ring closure, ρ-RNA interactions and ρ association with transcription elongation complexes. Structure-guided mutagenesis coupled with functional assays confirms that the observed ρ-Rof interface is required for Rof-mediated inhibition of cell growth and ρ-termination in vitro. Bioinformatic analyses reveal that Rof is restricted to Pseudomonadota and that the ρ-Rof interface is conserved. Genomic contexts of rof differ between Enterobacteriaceae and Vibrionaceae, suggesting distinct modes of Rof regulation. We hypothesize that Rof and other cellular anti-terminators silence ρ under diverse, but yet to be identified, stress conditions when unrestrained transcription termination by ρ may be detrimental.

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Introduction

Bacterial transcription termination factor, ρ, is a hexameric, NTP-dependent 5’–3’ RNA translocase and helicase. A ρ monomer contains N-terminal (NTD) and C-terminal (CTD) domains connected by a flexible linker¹. The NTD comprises an N-terminal three-helix bundle and a five-stranded β-barrel, the OB-fold, that forms a primary RNA-binding site (PBS); the PBSes are located around one outer rim of the ρ hexamer. The CTDs jointly form a secondary RNA-binding site (SBS) at the center of the ρ hexamer. The ligand-free ρ hexamer adopts an inactive, open-ring conformation, in which it can bind single-stranded RNA (or DNA) at the PBSes². When NTP (preferably ATP) binds to pockets located between the ρ subunits and RNA binds to the SBS, the ring closes, trapping RNA in the central pore of the hexamer and activating the ρ ATPase^3,4,5. Binding of pyrimidine-rich nucleic acids to the PBSes promotes the formation of a closed-ring active state⁶.

ρ triggers premature release of antisense, horizontally-acquired and untranslated RNAs from transcription complexes, ensuring that useless or potentially harmful RNAs are not transcribed¹. Productive RNA synthesis requires protection of transcription complexes from ρ. When RNA and protein synthesis are coupled, such as in fast-growing Escherichia coli, a translating lead ribosome is the main line of defense against ρ, as it denies ρ access to the transcribing RNA polymerase (RNAP) or nascent RNA⁷. Bacterial RNAs that are translated poorly (e.g., transcripts of xenogeneic cell wall biosynthesis operons) or not at all (e.g., ribosomal [r] RNAs) require dedicated anti-termination mechanisms to avoid being terminated by ρ¹. Bacteriophages, pervasive mobile elements that are silenced by ρ^8,9, use analogous mechanisms to ensure that their genes are expressed. Anti-termination is mediated by large ribonucleoprotein complexes, e.g., during rRNA synthesis or phage λ gene expression^10,11, single proteins such as RfaH^12,13 or RNAs^14,15,16, which are recruited to specific sequences or encoded within their target operons and inhibit termination within just one or a few operons.

It is currently unknown how ρ activity is regulated during periods of slow growth or stress, when translation is inefficient and uncontrolled transcription termination may be detrimental. Cellular levels of ρ are kept almost constant¹⁷ through auto-regulation¹⁸, implying that during stress, termination may be kept in check by regulators that reduce ρ activity. Three cellular anti-termination factors have been reported to inhibit ρ; the RNA chaperone Hfq, the Ser/Thr kinase YihE and the small protein YaeO (a.k.a. Rof in E. coli). Hfq can directly bind and inhibit ρ by an unknown, and possibly indirect, mechanism^16,19. YihE forms a complex with ρ and inhibits ρ-RNA interactions²⁰. Rof proteins adopt an Sm-like fold, resembling Hfq (Fig. 1a), and also directly bind ρ^21,22. Rof and bicyclomycin, an antibiotic that counteracts ρ ring closure⁶, have similar effects on the synthesis of a small RNA, sgrS²³. NMR-based docking and RNA binding studies suggested that Rof competes with RNA for binding to ρ²¹, but the precise mode of the ρ-Rof interaction and of Rof-mediated ρ inhibition remain unclear.

**Fig. 1: Rof structure, functions, conservation and genetic organization.**

In this work, we use a combination of structural, biochemical and genetic approaches to delineate in detail the mechanism of Rof-mediated inhibition of ρ. We show that Rof reduces ρ-dependent termination in vitro and inhibits E. coli growth, presumably by interfering with the essential functions of ρ. We present cryogenic electron microscopy (cryoEM)/single-particle analysis (SPA)-based structures of ρ-Rof complexes, precisely defining the ρ-Rof interfaces and showing that Rof undergoes substantial conformational changes upon binding to ρ. Substitutions of interface residues interfere with Rof activity in vitro and in vivo, confirming that the observed molecular interactions are required for ρ inhibition. Our structure of ρ bound to a 99-nt long RNA that concomitantly occupies the PBSes and the SBS confirms the existence of an extended PBS²⁴ that is blocked upon Rof binding. Furthermore, structural comparisons show that Rof undercuts functional communication between ρ NTDs and CTDs via the connecting linker. In addition, we show that Rof prevents ρ binding to a transcription elongation complex (EC). Thus, Rof blocks termination by inhibiting ρ recruitment to either the naked RNA or to ECs. We hypothesize that Rof and other known or yet to be identified anti-terminators mediate stress-specific responses by attenuating ρ activity.

Results

Rof is toxic in vivo and prevents ρ-dependent transcription termination in vitro

A pioneering study of E. coli (ec) Rof had shown that, when overexpressed, Rof co-purifies with ρ and induces readthrough of a canonical ρ terminator in the cell; surprisingly, however, purified Rof did not inhibit termination in vitro²⁵. We wondered if the presence of an N-terminal His-tag could have been responsible for this apparent loss of inhibition. The cellular function of Rof is not yet known, prompting us to establish a proxy assay for Rof activity in vivo. We previously showed that ectopic expression of Psu, an unrelated phage P4 inhibitor of ρ, directed by an IPTG-inducible P_trc promoter was toxic²⁶; we, thus, constructed a plasmid with the rof open reading frame (ORF) under the control of P_trc. A plasmid carrying wild-type (wt) rof, but not an empty vector, strongly inhibited growth of IA227²⁷, a derivative of MG1655 (Fig. 1b). Rof-mediated toxicity was abolished by the addition of an N-terminal His-tag or substitution of D14, a conserved Rof residue shown to be critical for the ρ-Rof interaction²¹, for alanine (Fig. 1b).

To determine at what stage of cellular growth Rof expression is inhibitory, we monitored the growth kinetics of IA227. We found that Rof^wt extended the lag phase from 2.75 to 12.65 h but did not alter the maximal growth rate or the saturation point (Supplementary Fig. 1a). The extended lag could reflect defects in protein synthesis required to sustain rapid growth^28,29 or loss of viability of the seed culture in the stationary phase. In support of the second explanation, Rof expression in IA227 strongly reduced viable colony count (Fig. 1b). By contrast, Rof expression was not toxic to wt E. coli and Salmonella typhimurium strains^23,30. To determine the cause of Rof toxicity in IA227, we sequenced its genome; we found that IA227 differs from the MG1655 reference genome in positions of mobile elements and several nucleotide polymorphisms (Supplementary Table 1). Among them is an early stop codon in rpoS, which encodes a “general stress response” σ^S factor that orchestrates adaptation to changes in cellular environment and adverse conditions³¹. Our results show that the rpoS null sensitizes cells to Rof (Supplementary Fig. 1b), suggesting an interplay between Rof activity and (still unknown) stress.

While our in vivo results (Fig. 1b) support the hypothesis that the failure to observe anti-termination in vitro²⁵ could be due to the presence of the His-tag on Rof, it is also possible that Rof, proposed to compete with RNA for binding to ρ²¹, may not be able to outcompete a tightly bound RNA; the failed in vitro trials²⁵ used λ tR1, one of the highest-affinity ρ ligands known. To test this idea, we compared effects of tag-less Rof in vitro on templates carrying λ tR1 and a leader region of Salmonella siiE, which is inhibited by ρ in vivo but lacks C-rich rut elements preferentially recognized by ρ³². We found that ρ terminated RNA synthesis on both templates (Fig. 1c), supporting a view that the rut site is less important in the context of the transcription complex^33,34. Tag-less Rof efficiently counteracted ρ-dependent termination on both templates, suggesting that an N-terminal His-tag indeed interferes with Rof activity and arguing that the Rof effects on ρ are direct and independent of rut sites (Fig. 1c).

Rof binds to ρ via a conserved interface

Consistent with pull-down assays²⁵, we found that Rof forms a stable complex with ρ in analytical size-exclusion chromatography (SEC), as a mixture of both proteins eluted at an earlier volume than either protein alone (Fig. 2a). Multi-angle light scattering (MALS) detection revealed that ρ and the ρ-Rof complex had heterogeneous molecular mass distributions (Fig. 2a), consistent with earlier observations that ρ does not exist as a discrete hexamer in solution³⁵.

We subjected SEC fractions corresponding to ρ-Rof complexes to cryoEM/SPA in the absence and presence of the transition state analog, ADP-BeF₃ (Supplementary Figs. 2–5 and Supplementary Table 2). Multi-particle 3D refinement with ~600,000 particle images yielded two cryoEM reconstructions for each sample (Supplementary Figs. 3 and 5), corresponding to two ρ-Rof assemblies. In the first assembly, ρ forms an open hexameric ring (ρ_A-ρ_F) bound by five Rof molecules (Rof_a-Rof_e; Fig. 2b), while the second assembly represents a ρ pentamer (ρ_A-ρ_E) bound by four Rof molecules (Rof_a-Rof_d). In the presence of ADP-BeF₃, we obtained a ~1:1 (ρ₆-Rof₅:ρ₅-Rof₄) distribution between the two classes, whereas in the absence of the nucleotide, the equilibrium was shifted towards the lower oligomeric assembly (ρ₆-Rof₅:ρ₅-Rof₄ ~ 1:3). The local resolutions of the reconstructions decrease towards the regions corresponding to terminal ρ subunits and bound Rof (Supplementary Figs. 2e and 4e). Weaker density for the peripheral Rof molecules could indicate a higher flexibility or a lower occupancy. We built an initial atomic model of ρ-bound Rof based on the density for Rof_c in the ρ₆-ADP-Rof₅ structure, which is best defined (Fig. 2b, c). This model was used as a starting model for other Rof molecules in the various complexes. The reconstructions do not reveal differences in the ρ-Rof interfaces, irrespective of oligomeric state or bound nucleotide. We therefore focus our subsequent descriptions on the ρ₆-ADP-Rof₅ complex and on Rof_c when discussing interaction details.

Rof molecules are bound between protomers ρ_A-ρ_B, ρ_B-ρ_C, etc., but not preceding or following the terminal ρ subunits, ρ_A and ρ_F (Fig. 2b). In each case, Rof interacts primarily with one ρ NTD through its N-terminal α-helix (α1, residues 9–23), the loop connecting strands β3 and β4 (residues 46–50) and the very C-terminal region (Fig. 2c). In addition, loop^β3-β4 of Rof contacts the N-terminal three-helix bundle of the adjacent ρ protomer (Fig. 2c), presumably explaining why Rof binding is only observed between neighboring ρ subunits but not at the terminal subunits of the open ring. The N-terminus and helix α1 of Rof are positioned in a cavity of the ρ NTD between ρ helix α4 (residues 83–89) and one flank of the ρ OB-fold (residues 95–120; Fig. 2c). Close contacts between the Rof N-terminus and ρ could explain why the addition of a His-tag blocks Rof activity in vivo (Fig. 1b). Loop^β3-β4 of Rof is positioned atop helix α5 within the ρ NTD-CTD linker (the “connector”) and the loop between helix α2 and α3 of the adjacent ρ protomer (residues 22–30; Fig. 2c). The very C-terminus of Rof rests on the exposed surface of the ρ OB-fold next to the Rof N-terminal region (Fig. 2c).

ρ-Rof contacts are predominantly polar (Fig. 2d–h and Supplementary Fig. 6). The backbone carbonyl oxygen of Rof^I8 (N-terminus) as well as the side chains of Rof^Y13 (helix α1) and Rof^E50 (β3-β4 loop) contact ρ^R88 by hydrogen bond, cation-π and ionic interactions, respectively (Fig. 2d). Rof^C10 and Rof^E17 (helix α1) form hydrogen bonds and a salt bridge with ρ^Q85 and ρ^R87, respectively (Fig. 2e, f). Rof^N48 (β3-β4 loop) is hydrogen bonded to ρ^R128 (connector; Fig. 2g). The C-terminal carboxy group of Rof^S84 approaches ρ^K100/K115 (Fig. 2h). Less well-defined interactions include Rof^D14 (helix α1) hydrogen bonding with ρ^S82/S84 (Fig. 2e) and Rof^K47 (β3-β4 loop) interacting electrostatically with ρ^E24 of the adjacent ρ subunit (Fig. 2g). Most of the ρ-contacting residues of Rof are conserved (Supplementary Fig. 6a). Likewise, Rof-contacting residues of ρ are highly conserved (Supplementary Fig. 6b). As ρ is more widespread among bacteria than Rof, the conservation of the Rof-contacting ρ residues likely reflects their importance in forming the PBS.

Replacement of ρ-contacting residues undermines Rof function

To validate our structural observations, we replaced Rof residues Y13, D14 or E17 in helix α1 with alanine and tested binding of the corresponding Rof variants to ρ^wt in analytical SEC (Fig. 3a). While the ρ interaction was only very mildly affected by an E17A exchange in Rof, ρ binding was completely abolished in Rof^Y13A and Rof^D14A, as monitored by SEC. We also generated ρ variants, ρ^R88E, ρ^F89S and ρ^K115E, and tested them for Rof^wt binding (Fig. 3a). Interaction of ρ^R88E with Rof^wt was abrogated in SEC, while Rof^wt binding was only slightly reduced in ρ^F89S or ρ^K115E (Fig. 3a). Thus, our structures delineate a precise network of interactions that are crucial for ρ-Rof complex formation.

**Fig. 3: Effects of Rof and ρ variants.**

Rof variants that were unable to bind ρ were also unable to elicit anti-termination in vitro: ρ terminated synthesis of 90% and 86% of transcripts in the presence of Rof^Y13A and Rof^D14A, respectively, as compared to 16% in the presence of Rof^wt and 87% in the absence of Rof (Fig. 3b). In agreement with only slightly reduced affinity of Rof^E17A for ρ, Rof^E17A was only slightly less effective in inhibiting ρ than Rof^wt (25% of transcripts terminated in the presence of Rof^E17A; Fig. 3b). These results indicate that the ρ-Rof contacts observed in our structures are relevant for Rof-dependent inhibition of ρ in the context of complete ECs.

Next, we evaluated the effects of exchanging Y13, D14 and E17 and an additional ρ-contacting residue in helix α1 of Rof, C10, on toxicity in vivo. We measured the lag phase extension upon IPTG induction of plasmid-encoded Rof variants (Fig. 3c). While Rof^wt increased the lag 4.6–fold, substitutions of several conserved residues in the N-terminal helix of Rof strongly reduced this effect: plasmids guiding the production of Rof^Y13A, Rof^D14A or N-terminally His-tagged Rof were indistinguishable from the empty vector, whereas Rof^C10S (Fig. 2e) increased the lag 2.4-fold. As Rof^E17A did not show significant defects in vitro (Fig. 3b), we tested the effects of the Rof^E17K variant instead; the charge reversal led to a complete loss of Rof toxicity. By contrast, substitutions in the Rof β3-β4 loop (Fig. 2g) had smaller effects: R46A and K47A exchanges in Rof increased the lag 2.6- and 3.2-fold, respectively, and Rof^N48A was almost indistinguishable from Rof^wt (Fig. 3c). We conclude that in particular contacts between ρ and the N-terminal helix of Rof observed in our structures are important for Rof-mediated inhibition of E. coli growth.

Substitutions of the surface-exposed Rof residues that contact ρ would not be expected to alter the Rof structure, and thus cellular production or stability. To ascertain that the observed in vivo defects are due to the loss of binding of the Rof variants to ρ, we used Western blotting to quantify Rof variants in cells. As specific anti-Rof antibodies are not yet available, we inserted a single HA tag into a flexible loop of Rof at residue 57, the only location that we found to tolerate tags without the loss of in vivo activity. We found that D14A, E17K, R46A and E47A variants of Rof were produced at levels comparable to, or higher than, Rof^wt, whereas the Rof^Y13A variant was less abundant (Fig. 3d).

Rof undergoes pronounced conformational changes upon binding to ρ

A solution structure (PDB ID: 1SG5)²¹ and an AlphaFold³⁶ model of isolated E. coli Rof showed marked differences and high flexibility in the N-terminal region compared to our ρ-bound Rof. In the isolated state, the N-terminal region lacks contacts to the globular portion of Rof and thus remains flexible, while it is immobilized and caps one face of the OB-fold in the ρ-bound state (Fig. 4). Concomitantly, helix α1 is oriented differently relative to the β-barrel in the isolated and ρ-bound states (Fig. 4). As a consequence, the N-terminal region and helix α1 are repositioned relative to the other main ρ-interacting regions of Rof, the β3-β4 loop and the C-terminus (Fig. 4). The above comparisons show that, upon ρ engagement, Rof undergoes conformational changes and folding/immobilization transitions, which are required to generate the productive ρ-binding surface, composed of N-terminal region, helix α1, β3-β4 loop and C-terminus. In contrast to Rof, no global conformational changes are observed in the ρ protomers upon Rof engagement (e.g. compared to an open-ring, ANPPNP-bound ρ structure; PDB ID: 1PVO)².

**Fig. 4: Rof conformational changes upon ρ binding.**

A model of an E. coli ρ-Rof complex has previously been proposed based on the solution structure of Rof and NMR-guided docking²¹. While the general binding site of Rof on the ρ NTDs agrees with our cryoEM structures, the previous docking model differs significantly in detail, as the conformational changes upon binding were not captured. Presently, we cannot exclude that a minor population of isolated Rof adopts a conformation observed in the ρ-bound state, and that binding might, thus, occur via a conformational selection mechanism. However, presently available data favor a mechanism that involves binding via a pronounced induced fit in Rof. Irrespectively, apart from steric hindrance (see above), an N-terminal tag on Rof might also interfere with the conformational changes and folding/immobilization upon binding required for stable complex formation with ρ, as also observed with other proteins³⁷.

Rof blocks an extended PBS of ρ

Electrophoretic mobility shift assays indicated that Rof interferes with RNA binding to the ρ PBSes²¹. To further evaluate Rof effects on RNA binding at ρ PBSes, we quantified the affinity of a 5’-FAM-labeled DNA oligomer, dC₁₅, that binds exclusively to the ρ PBS⁶ by fluorescence anisotropy (Fig. 5a). ρ alone bound dC₁₅ tightly (K_d = 1 µM; Fig. 5b), in agreement with previous results³⁸. ρ binding to dC₁₅ was reduced stepwise in the presence of increasing concentrations of Rof^wt, but not in the presence of Rof^Y13A or Rof^D14A (Fig. 5b), suggesting that Rof blocks the ρ PBSes or parts thereof.

**Fig. 5: Effect of Rof on RNA binding by ρ.**

A structure of ρ bound to a short oligonucleotide at the PBS (PDB ID: 1PVO) had delineated a core PBS that accommodates a dinucleotide², and comparison to our ρ-Rof complex structures showed that Rof does not interfere with RNA contacts at the core PBS. However, a recent structure of a pre-termination complex showed that RNA can additionally occupy a region proximal to the core PBS²⁴, consistent with the observation that longer PBS ligands bind ρ more tightly and have greater impact on ρ ring closure^3,6. We attempted to further narrow down the path of RNA along the ρ PBSes by determining a cryoEM/SPA structure of ρ in complex with a 99-nt long, natural rut RNA derived from the λ tR1 terminator, which encompasses both SBS- and PBS-binding regions (Supplementary Fig. 7a). In the presence of ADP-BeF₃, we obtained one reconstruction at a global resolution of 2.9 Å (Supplementary Figs. 8 and 9 and Supplementary Table 2).

In the ρ-rut RNA structure, ρ adopts a closed conformation very similar to ρ in complex with an rU₇ SBS ligand and ADP-BeF₃ (PDB ID: 5JJI³) and to ρ in a pre-termination complex (PDB ID: 8E6W²⁴; Fig. 5c). Density for six nucleotides (nts) is clearly defined at the SBS in the center of the ring (Supplementary Fig. 7b). We tentatively assigned the sequence at the very 3’-end of the RNA ligand to this region (Supplementary Fig. 7a). Density corresponding to RNA regions at the ρ PBSes is fragmented, suggesting that corresponding RNA regions are dynamically bound and precluding their reliable modeling. Irrespectively, the reconstruction is consistent with six nts bound in an extended conformation (Fig. 5d). No density is observed connecting the PBS-bound RNA regions or between a PBS-bound RNA region and RNA at the SBS (Fig. 5c). At the PBSes, the two 3’-most nts are accommodated at the core PBS as observed before³. Density for the preceding nts is lined by ρ^K102, ρ^R105 and ρ^K115 on one side, and by ρ^D60, ρ^F62, ρ^P83, ρ^S84, ρ^Q85, ρ^R87, ρ^R88 and ρ^F89 on the other (Fig. 5e). Consistent with this observation, binding of ρ^R88E and ρ^K115E variants to the λ tR1 rut RNA was completely abolished or significantly reduced, respectively, compared to ρ^wt, while binding of ρ^F89S was undisturbed (Supplementary Fig. 7c). These results confirm and further validate an “extended PBS” as observed in a pre-termination complex²⁴, which augments the RNA affinity and specificity of the ρ core PBS.

Structural comparisons between our ρ-Rof and ρ-rut RNA structures showed that bound Rof would block RNA binding at the extended PBS (Fig. 5f). Consistent with the idea that Rof blocks ρ activity by outcompeting RNA at the ρ extended PBS, residues that contribute to the extended PBS are also important for Rof binding (Figs. 2d–h and 5e).

RNA binding at the extended PBS supports ρ dynamics accompanying ring closure

The classical mechanism of ρ-dependent termination³⁹, recently visualized by cryoEM²⁴, involves ATP hydrolysis-dependent 5’–3’ translocation of ρ on RNA. To activate ATP hydrolysis and RNA translocation, ρ has to convert from an open to a closed-ring conformation^3,4. Ring closure involves conformational changes in ρ that affect the protomer interfaces^3,5. Upon ring closure, the NTDs of adjacent ρ subunits separate, breaking contacts between the NTD-CTD connector of one ρ protomer and the N-terminal three-helix bundle of the adjacent ρ protomer; simultaneously, the ρ CTDs approach each other more closely, encircling the RNA at the SBS and forming ATPase-active ATP-binding pockets^2,5.

In open ρ structures, ρ^R128 at the C-terminus of connector helix α5 contacts ρ^N25 of a neighboring NTD, helping to position ρ^R28 of the adjacent protomer in a binding pocket that involves helix α5 of the connector (Fig. 6a). These inter-subunit contacts are broken during ring closure; ρ^R128 now hydrogen bonds to the backbone carbonyl of ρ^R88 and/or is engaged by the side chain of ρ^E125, and ρ^R28 is exchanged for ρ^K130 of the same protomer (Fig. 6b). Furthermore, in open and closed ρ structures, the NTD-CTD connector establishes different contacts to the NTD and CTD of its own protomer as well as to the neighboring ρ subunit; upon ring closure, interactions along the entire NTD-CTD connector shift in register by one residue^40,41 (Fig. 6c, top and middle panels). Thus, the NTD-CTD connector establishes dynamic interaction networks, differentially interconnecting the NTD and the CTD within a protomer as well as differentially contacting the neighboring protomer in the open and closed states.

**Fig. 6: Effects of Rof on ρ conformation.**

Apart from ATP binding between neighboring CTDs and RNA binding at the SBS, ring closure is also promoted by RNA binding at the PBSes^27,41. RNA binding at the extended PBS observed in a pre-termination complex²⁴ and in our ρ-rut structure could explain this phenomenon. Residues forming the extended PBS and the 5’-end of the PBS-bound RNA region are in direct proximity of connector helix α5. We surmise that RNA bound at the extended PBS supports the closed-ring interaction network by structurally stabilizing the NTD, reinforcing the sequestration of ρ^R128 and the repositioning of ρ^K130 (Fig. 6b).

Rof stabilizes the open ρ conformation and undercuts connector-mediated communication between ρ domains

Upon binding, Rof positions its β3-β4 loop on top of helix α5, establishing direct contacts of Rof^N48 to ρ^R128 and thus reinforcing the interaction of ρ^R128 with ρ^N25 of the neighboring NTD (Fig. 2g). Furthermore, Rof^K47 in the β3-β4 loop engages in a salt bridge with ρ^E24 of the neighboring ρ protomer (Fig. 2g). Thus, Rof bound between two neighboring NTDs of open ρ may stabilize their relative positions, and thus the open-ring conformation. To test whether Rof indeed counteracts ρ ring closure, we performed fluorescence anisotropy assays with the 5’-FAM-labeled SBS ligand, rU₁₂, in the presence of unlabeled dC₁₅ that blocks the PBSes⁶, such that rU₁₂ binding at the SBS reports on ρ ring closure (Fig. 5a). Indeed, while ρ alone readily underwent ring closure, Rof inhibited ring closure in a concentration-dependent manner (Fig. 5g).

Surprisingly, although ρ adopts an open conformation upon Rof binding, the connector retains the NTD/CTD-interaction register of the closed conformation (Fig. 6c, middle and bottom panels), presumably due to the direct Rof-helix α5 interactions restricting connector rearrangements. Concomitantly, in complex with Rof, the CTD Q-loops adopt a conformation observed in closed ρ, but not in open ρ structures in the absence of Rof ⁴² (Fig. 6d); in the closed ring as well as in the open ring in complex with Rof, ρ^K283 is inserted into a pocket formed by the Q-loop of the adjacent protomer, while ρ^K283 points to the center of the ring in open ρ in the absence of Rof (Fig. 6d).

Together, these observations show that Rof effectively stabilizes ρ in an open conformation by cross-strutting neighboring NTDs and preventing their separation, yet at the same time prevents a register shift and change in interactions along the NTD-CTD connector, maintaining the CTDs in a conformation resembling the closed state. Thus, Rof conformationally insulates ρ NTDs and CTDs by undercutting concerted structural transitions normally associated with ρ ring dynamics and induces a hybrid conformation, with the NTDs in the open state and the connector and CTDs in the closed-state conformation.

Rof interferes with ρ binding to transcription elongation complexes

Recent structural studies had shown that ρ can engage ECs modified by general transcription factors NusA and NusG before binding the transcript, with the ρ NTDs establishing direct contacts to NusA, NusG and RNAP subunits α, β and β’^33,34. In the ρ-bound NusA/NusG-ECs, ρ remains dynamic relative to the surface of RNAP, allowing ρ to position one of its PBSes next to the RNA exit channel to eventually capture the nascent transcript. Subsequent conformational changes in RNAP can trigger transcription inactivation.

Superposition of the ρ-Rof and ρ-EC structures revealed that ρ surfaces in contact with ECs encompass the extended PBSes, and that Rof binding to ρ is incompatible with ρ binding to an EC (Fig. 7a). Consistently, when in complex with Rof, ρ showed impaired binding to a NusA/NusG-EC in analytical SEC (Fig. 7b; compare panels 2 and 3). Similarly, ρ dissociates from a pre-formed ρ/NusA/NusG-EC upon addition of Rof (Fig. 7b; compare panels 2 and 4), indicating that Rof can act on ρ-bound transcription complexes within the cell. Thus, in addition to preventing productive engagement of a transcript, Rof also hinders EC engagement by ρ.

**Fig. 7: Rof effects on ρ-EC interactions.**

Rof proteins from Vibrionaceae may resort to a modified ρ-inhibitory mechanism

Unlike ρ, which is ubiquitous in bacteria, Rof is restricted to Pseudomonadota (synonym Proteobacteria; Fig. 1d); 79% of Enterobacteriaceae and 63% of Vibrionaceae genomes are estimated to encode Rof (Supplementary Data 1). In E. coli, rof is encoded in a five-gene locus bracketed by REP elements (Fig. 1e). Two genes in this cluster, arfB and nlpE, play vital roles in E. coli stress response. ArfB, a peptidyl-tRNA hydrolase, rescues stalled ribosomes when trans-translation control fails⁴³. NlpE is an outer membrane lipoprotein that activates the Cpx pathway in response to envelope stress⁴⁴. Rof is expressed from a divergent promoter in tandem with a small conserved protein of unknown function, YaeP⁴⁵.

Presence of rof in diverse Pseudomonadota (Fig. 1d) suggests a common mechanism of ρ inhibition. However, recombinant V. cholerae Rof (vcRof) has been observed to form higher-order oligomers in vitro, of which the dimer state could be stabilized by treatment with iodoacetamide/TCEP; furthermore, vcRof seems to disassemble ρ hexamers in vitro²². ρ-bound ecRof and a vcRof monomer (PDB ID: 6JIE)²² exhibit similar structures that superimpose with a root-mean-square deviation (rmsd) of 1.05 Å for 58 pairs of Cα atoms (2.65 Å for all 72 pairs of Cα atoms). Similar to isolated ecRof, isolated vcRof differs from ρ-bound ecRof in the orientation of the N-terminal region, which is diverted from the OB-fold; furthermore, helix α1 in isolated vcRof is shortened by one turn compared to ecRof (Fig. 8a, b). Given that crucial ρ-contacting residues in the N-terminal regions are highly conserved between ecRof and vcRof (Fig. 8c), vcRof most likely binds its cognate ρ in a similar manner, undergoing conformational changes as revealed here for ecRof. However, more pronounced sequence divergence in other regions of Rof proteins from Enterobacteriaceae and Vibrionaceae (Fig. 8c), including in the ρ-contacting β3-β4 loops (Fig. 2g), may reflect some differences in the specific contacts to ρ.

**Fig. 8: Rof conformational changes and conservation.**

A heterotypic configuration of the vcRof dimer has been proposed based on a crystal structure (PDB ID: 6JIE)²². While putative ρ-interacting surfaces, in particular the N-terminal region, of one vcRof subunit are involved in the proposed dimerization, the putative ρ-interacting elements of the other vcRof subunit are unobstructed; thus, while the proposed vcRof dimer could engage ρ via one of the vcRof subunits as observed for our E. coli ρ-Rof complexes without steric hindrance (Fig. 8d, top), conflicts would ensue upon dimeric vcRof binding to ρ via the subunit with partially buried ρ-interacting elements (Fig. 8d, bottom). While such engagement may in principle lead to conformational changes in ρ and disassembly of the hexamer, the molecular mechanisms that would favor this disruptive engagement over binding of dimeric vcRof via the unobstructed subunit remain unclear.

Apart from differences in the interaction details and the oligomeric states, other factors may differentially influence the mode of action of Rof proteins in Enterobacteriaceae and Vibrionaceae. We observed that the rof genomic contexts in Enterobacteriaceae and Vibrionaceae are strikingly different. While in Enterobacteriaceae, the rof gene always overlaps with an upstream yaeP gene, the yaeP and rof genes are located on different chromosomes in V. cholerae (Fig. 8e and Supplementary Data 1). Furthermore, rof does not have conserved gene neighbors in Vibrionaceae (Supplementary Fig. 10 and Supplementary Data 1). Collectively, our analyses reveal that monomeric ecRof and dimeric vcRof could engage ρ in an analogous fashion and thus inhibit ρ function via similar molecular principles. The molecular mechanisms of putative additional levels of ρ inhibition via subunit dissociation by vcRof as well as possible differences in Rof regulation remain to be elucidated.

Discussion

ρ is a global gene regulator and is essential in most bacterial species. It defines gene boundaries, silences production of useless and harmful RNA, and resolves R-loops¹. Single-molecule fluorescence studies are consistent with the idea that ρ-dependent termination can proceed via two pathways^46,47. In the classical RNA-dependent pathway, ρ binds C-rich, unstructured and unoccupied RNA regions via its PBSes; subsequent binding of a neighboring RNA region at the SBS and ring closure allow ρ to translocate towards elongating RNAP, eventually contact the transcriptional machinery around the RNA exit channel and terminate transcription^24,48. In the EC-dependent pathway, which may also represent a prelude to ultimate termination via the classical mechanism³³, ρ associates with and accompanies ECs without immediately terminating transcription⁴⁹; upon EC pausing, NusA and NusG can cooperate with ρ in inducing conformational changes in RNAP that stall transcription before ρ engages the transcript^33,34.

A deleterious mutation in rho enables E. coli survival during proteotoxic stress⁵⁰ and envelope stress turns on the expression of YihE, a Ser/Thr kinase that inhibits ρ/RNA interactions²⁰. These findings suggest that ρ activity may need to be tuned down under some stress conditions. The RNA chaperone Hfq, which has been implicated in diverse stress responses⁵¹, inhibits ρ function^16,19 as does another Sm-like protein, Rof ²⁵, but their mechanisms remained incompletely understood. Here we present structural, functional and phylogenetic analyses of ρ regulation by E. coli Rof, which are in agreement with a study of Salmonella Rof by Zhang et al. ³⁰.

Extending and detailing an earlier docking model²¹, our results show that Rof undergoes pronounced conformational changes and folding/immobilization upon binding to the NTDs of an open ρ ring, thereby implementing multiple strategies to counteract both ρ-dependent termination pathways. First, Rof prevents ρ from binding to RNA at the PBS by blocking an extended PBS. An extended PBS has long been envisioned to contribute to both the RNA affinity and specificity of ρ, even though early structures of open ρ revealed only a dinucleotide bound at the core PBS (PDB ID: 1PVO)² and early structures of closed ρ lacked RNA at the PBS (PDB ID: 3ICE)⁵. A recent structure of a ρ pre-termination complex²⁴ disclosed RNA bound along extended PBSes and at the SBS of a closed ρ ring, as confirmed by our ρ-rut RNA structure. We show that ρ residues contacting Rof are also crucial for binding RNA at the extended PBS (Fig. 5d–f and Supplementary Fig. 7c) and are highly conserved (Supplementary Fig. 6b).

Second, Rof prevents ρ ring closure and thus RNA binding at the SBS (Fig. 5g). Given that optimal SBS ligands (e.g., rU₁₂) promote ring closure even in the absence of a PBS ligand⁶, PBS blocking alone cannot fully accounts for Rof-mediated inhibition of ρ ring closure. Furthermore, as Rof itself binds at the ρ PBSes—why does Rof binding not induce ring closure? Our results show that Rof bridges the NTDs of two adjacent ρ protomers, thus preventing their separation that is required for ring closure (Fig. 2c, g).

Third, Rof conformationally insulates the ρ NTDs and CTDs, undercutting conformational dynamics involved in RNA binding at the SBS and ring closure. Functional studies had suggested that residues forming the core and extended PBSes are involved in an allosteric network that mediates communication across ρ domains via the NTD-CTD connector. For example, a Y80C substitution in the NTD alters the CTD conformation⁴⁰, and ρ^R88 has been suggested to affect steps other than RNA binding at the PBS⁵². Our ρ-rut RNA structure suggests that such communication involves stabilization of the bound regions, which strengthens contacts of the NTD-CTD connector characteristic of the closed state. In addition, the 5’-end of PBS-bound RNA could directly contact the connector helix α5 and stabilize the closed-state configuration. Rof, in contrast, induces a chimeric structure in ρ wherein NTDs adopt an open-ring arrangement, while the NTD-CTD connector and the CTDs resemble the closed state, presumably due to Rof also directly contacting helix α5 of the connector, thus stabilizing the closed-state register (Fig. 6c). In particular, the CTD Q-loops adopt a conformation very similar to that observed in closed-ring structures of ρ, with ρ^K283 embedded in a pocket formed by the Q-loop of the neighboring protomer (Fig. 6d). In open ρ complexes in the absence of Rof, ρ^K283 side chains instead point to the ring interior (PDB ID: 6WA8)⁴². We speculate that ρ^K283 in open ρ might be important for the initial RNA capture at the SBS and its proper alignment during ring closure. Thus, by retaining CTDs in a state resembling the closed ring, and thereby sequestering ρ^K283, Rof might also counteract initial RNA engagement at the SBS.

Fourth, as the extended PBSes and other Rof-contacting surfaces on ρ also represent main contact points of ρ on NusA/NusG-modified ECs^33,34, Rof interferes with ρ binding to ECs and can partially dissociate ρ from pre-formed ρ-ECs (Fig. 7). As Rof binding at the ρ PBSes would, thus, equally affect both, the RNA-dependent and EC-dependent termination pathways, our results do not favor one over the other.

Rof-mediated inhibition of ρ complements other strategies of ρ-regulation in bacteria (Fig. 9). Maintaining a balance between inactive ρ sequestered by Rof and unobstructed ρ that could engage nascent transcripts or ECs would allow cells to tune ρ-dependent transcription termination in response to cellular cues, e.g., during slow growth or under stress. Consistently, we found that the absence of σ^S, a master regulator of general stress response, sensitizes E. coli to Rof expression (Supplementary Fig. 1b). At least two key questions concerning the cellular role of Rof presently remain unanswered: when does the cell need Rof and how is Rof expression controlled? Rof is present in many Pseudomonadota, implying that its ability to dampen ρ-dependent termination is important under some conditions. A recent study showed that while the rof deletion did not alter growth of Salmonella in standard laboratory conditions, it attenuated virulence by suppressing the expression of genes encoded on the SPI-I pathogenicity island³⁰.

**Fig. 9: ρ regulation under optimal growth and stress conditions.**

As ρ is essential, a global anti-terminator should be produced in substantial amounts only when needed. Indeed, a quantitative proteomic analysis of E. coli¹⁷ demonstrated that both Rof and YihE are present at low (and similar) levels across many conditions, with a modest increase during the stationary phase, consistent with the first report on Rof ²⁵. YihE is activated by Cpx⁵³, a two-component system that responds to envelope stress⁴⁴. Collectively, it stands to reason that the expression of yaeP-rof is induced by particular cellular signals; e.g., a modest increase in the yaeP-rof transcript level was observed during recovery from exposure to mercury⁵⁴. Identification of other triggers awaits a systematic analysis. Bacteria experience a wide variety of adverse conditions and inhibiting ρ could be essential for survival in some, but not other, situations. We hypothesize that Rof and YihE belong to a large set of specialized anti-termination factors, which could comprise proteins and sRNAs, that turn off ρ under diverse stress conditions to enable survival and facilitate recovery (Fig. 9).

Methods

Recombinant protein and RNA production and purification

Supplementary Table 3 lists plasmids used in this study. E. coli ρ protein and variants thereof were produced and purified as described before¹¹. DNA encoding Rof was codon optimized for E. coli expression and purchased from Invitrogen (Thermo Fisher Scientific). The rof gene was cloned into pETM-11 and expressed in E. coli RIL (BL21) cells (Novagen). Cells were lysed by sonification in buffer A (100 mM KCl, 5 mM MgCl₂, 20 mM Na-HEPES, pH 7.5, 1 mM DTT, 10% [v/v] glycerol, 1 mM DTT) and the lysate was cleared by centrifugation at 55,914 x g for 1 h at 4 °C. The cleared lysate was loaded on a 5 ml Ni²⁺-NTA column (Cytiva) equilibrated in buffer A. Rof was eluted in elution buffer (buffer A supplemented with 500 mM imidazole). The eluate was treated with TEV protease and dialysed overnight against buffer A with 5% (v/v) glycerol. After recycling on the Ni²⁺-NTA column, the flow-through was collected, concentrated and loaded to a Superdex 75 column (Cytiva) equilibrated in SEC buffer (100 mM KCl, 5 mM MgCl₂, 20 mM Na-HEPES, pH 7.5, 1 mM DTT). Peak fractions were pooled and concentrated to 4.4 mM. Rof was flash frozen in liquid nitrogen and stored at -80 °C until further use. rho and rof mutations were introduced by site directed mutagenesis using the QuikChange protocol (Stratagene) and protein variants were produced and purified as for the wt versions. In vitro transcribed rut RNA was produced and purified as described before³³.

SEC-MALS

SEC-MALS was performed in reaction buffer (20 mM Tris-HCl, pH 8.0, 120 mM KOAc, 5 mM Mg(OAc)₂, 2 mM DTT). 1 mg/ml of ρ were mixed with 2 mg/ml of Rof and incubated at 32 °C for 10 min. 60 µl of the mixture were loaded on a Superose 200 increase 10/300 GL column (Cytiva) and chromatographed on HPLC system (Agilent) coupled to a miniDAWN TREOS multi-angle light scattering and a RefractoMax 520 detector system (Wyatt Technologies). Prior to measurements, a system calibration was performed using BSA (Sigma-Aldrich). ASTRA 6.1 software was used for data analysis and molecular mass determination.

Sample preparation for cryoEM

For ρ-Rof complex formation, 41.8 µM ρ hexamer were mixed with a 10-fold molar excess of Rof in the absence or presence of 2.3 mM ADP-BeF₃ in reaction buffer and incubated for 15 min at 32 °C. The mixture was applied to a Superdex 200 Increase 3.2/300 column (Cytiva) and fractions of the complex were pooled and concentrated. 3.8 µl of the purified complex (5.3 mg/ml) were applied to glow-discharged Quantifoil R1.2/1.3 holey carbon grids and plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher) set at 10 °C and 100% humidity.

For ρ-rut RNA complex formation, 41.8 µM ρ hexamer were mixed with an equimolar amount of rut RNA (Supplementary Table 3) and 2.3 mM ADP-BeF₃ in reaction buffer and incubated for 15 min at 32 °C. The mixture was applied to a Superose 6 Increase 3.2/300 column (Cytiva) and fractions of the complex were pooled and concentrated. Purified complex (3.8 mg/ml) was vitrified as above.

CryoEM data acquisition and processing

CryoEM data were acquired on a FEI Titan Krios G3i TEM operated at 300 kV equipped with a Falcon 3EC direct electron detector (EPU, version 2.8.1; Thermo Fisher Scientific). Movies for ρ-Rof samples were taken for 40.57 s accumulating a total electron dose of ~ 40 e^-/Å² in counting mode distributed over 33 fractions at a nominal magnification of 96,000 x, yielding a calibrated pixel size of 0.832 Å/px. Data for the ρ-rut RNA sample were acquired at a higher nominal magnification of 120,000 x, corresponding to a calibrated pixel size of 0.657 Å/px. A total electron dose of 40 e^-/Å² was accumulated over an exposure time of 30.58 s.

All image analysis steps were done with cryoSPARC (version 4.1.1)⁵⁵. Movie alignment was done with patch motion correction, CTF estimation was conducted with Patch CTF. Class averages of manually selected particle images were used to generate an initial template for reference-based particle picking from 4216 micrographs for the ρ-Rof sample. 1,782,459 particle images were extracted with a box size of 384 px and Fourier-cropped to 96 px for initial analysis. Reference-free 2D classification was used to select 1,101,769 particle images for further analysis. Ab initio reconstruction was conducted to generate an initial 3D reference for heterogeneous 3D refinement. 600,888 particle images were further classified by 3D variability analysis into ρ₆-Rof₅ and ρ₅-Rof₄ complexes. Particle images were re-extracted with a box size of 384 px after local motion correction and subjected to non-uniform refinement giving reconstructions at ~ 2.9 Å resolution each that could be slightly improved by CTF refinement. Another iteration of heterogeneous 3D refinement was applied to determine the final set of 110,264 particle images for the ρ₆-Rof₅ complex and 293,352 particle images for the ρ₅-Rof₄ complex. Non-uniform (NU) refinement yielded reconstructions at a global resolution of 2.92 Å and 2.74 Å, respectively. Data analysis for the ρ-Rof samples was conducted similarly.

Model building, refinement and analysis

Coordinates of ρ (PDB ID: 6WA8 [open ring]; PDB ID: 1PVO [closed ring]) and Rof (PDB ID: 1SG5) were docked into the cryoEM maps using Coot (version 0.8.9)⁵⁶. ρ subunits and Rof were manually adjusted to fit the cryoEM density. The rut RNA regions were built de novo except for the dinucleotide at the core PBS (taken from PDB ID: 1PVO)². Manual model building alternated with real space refinement in PHENIX (version 1.20.1)⁵⁷. Data collection and refinement statistics are provided in Supplementary Table 2. Structure figures were prepared with ChimeraX (version 1.6.1)⁵⁸.

Analytical size-exclusion chromatography

Proteins and/or nucleic acids were mixed in reaction buffer and incubated for at 32 °C for 10 min. 50 µl of the samples were loaded on a Superdex S200 P.C 3.2 column (ρ-Rof) or Superose 6 Increase 3.2/300 column (ρ-rut RNA) and chromatographed at a flow rate of 50 µl/min at 4 °C. To test for ρ-Rof interaction, 5 µM ρ and 90 µM Rof/variants were used. To test for ρ-rut RNA interaction, 4 µM ρ/variants and 5 µM rut RNA were used.

For testing ρ binding to ECs in the absence or presence of Rof, ECs composed of RNAP, NusA, NusG, nucleic acid scaffold containing a short rut-less RNA (16 nts) without or with ρ were formed as described before³³. 2.4 µM EC lacking ρ were incubated with a 3-fold molar excess of ρ-Rof complex at 32 °C for 15 min. Alternatively, 1.1 µM ρ-EC were incubated with a 10-fold molar excess of Rof at 32 °C for 15 min. Fractions were analyzed by SDS-PAGE and urea PAGE to reveal protein and nucleic acid contents, respectively. DNAs and RNAs used for complex assembly are listed in Supplementary Table 3.

Nucleic acid binding assays

Nucleic acid binding to ρ PBS or SBS was tested by fluorescence depolarization-based assays⁶. For PBS binding, 5 µM 5’-FAM-labeled dC₁₅ oligo (Eurofins) were mixed with increasing amounts of ρ (0 to 17 µM final hexamer concentration) in 20 mM Na-HEPES, pH 7.5, 150 mM KCl, 5% (v/v) glycerol, 5 mM MgCl₂, 0.5 mM TCEP. For SBS binding, ρ PBSes were first saturated with 10 µM non-labeled dC₁₅. ρ (0 to 16 µM final hexamer concentration) was then mixed with 2 mM ADP-BeF₃ and 2 µM 5’-FAM-labeled rU₁₂ oligo (Eurofins). To test the effect of Rof and Rof variants on nucleic acid binding to ρ PBS or SBS, 1 or 5 µM of ρ hexamer were mixed with increasing amounts of Rof (0 – 100 µM) prior to dC₁₅ addition. The fluorescence anisotropy was recorded in OptiPlateTM 384-well plates (PerkinElmer) using a Spark Multimode Microplate reader (Tecan; excitation wavelength, 485 nm; detected emission wavelength, 530 nm). Two technical replicates were averaged for each sample and the data were analyzed with Prism software (version 9.0.2; GraphPad). To quantify ρ PBS or SBS binding, data were fitted to a single exponential Hill function; Y = Y_max[protein]^h / (K_d^h + [protein]^h); Y_max, fitted maximum of nucleic acid bound; K_d, dissociation constant; h, Hill coefficient.

Plating efficiency assays

Individual colonies of each strain, in 3 biological replicates, transformed with plasmids carrying inserts of interest under the control of P_trc promoter were grown overnight in LB supplemented with carbenicillin at 32 °C. Serial dilutions of overnight cultures were spotted onto LB plates containing carbenicillin with and without IPTG.

Growth assays

Individual colonies of each strain, in 4 biological replicates, were inoculated in MOPS EZ rich defined media (Teknova, #M2105) supplemented with carbenicillin (100 mg/l) and grown overnight at 32 °C. The overnight culture was diluted (1.5 μl into 98.5 μl) in fresh media supplemented with 1 mM IPTG, loaded into a 96-well plate, and grown in a BioTek EPOCH2 microplate reader at 32 °C under control of Gen5 software (version 3.12; BioTek). The OD₆₀₀ in each well was measured in 15 min intervals for 24 h with kinetic shaking and incubation. Results were analyzed in Microsoft Excel.

Western blotting

Overnight cultures of cells transformed with plasmids encoding Rof variants were grown in LB + carbenicillin (100 mg/l) at 32 °C. After 1:100 dilution into fresh media, the cultures were grown to an early exponential phase at 32 °C, induced with 1 mM IPTG for 60 min, pelleted and frozen. Cell pellets were resuspended in PBS, sonicated and centrifuged. Protein concentration in cleared lysates was measured with Bradford reagent and normalized before resolving in SurePAGE Bis-Tris 8-16% gels (GenScript). Molecular weight markers were run on every gel to monitor separation. Proteins were transferred to a nitrocellulose membrane (Bio-Rad Trans-Blot Transfer Medium, Cat. 162-0112). Membrane was incubated with primary anti-HA antibodies from mice (1:5,000 dilution; Sigma-Aldrich, Cat. H9658) and with secondary anti-mouse horseradish peroxidase linked antibodies (1:10,000 dilution; Amersham, Cat. NA931V) before imaging using Bio-Rad Clarity Max Western ECL Substrate (Cat. 1705062 S) with a Bio-Rad ChemiDoc XRS+ system controlled using ImageLab (version 6.1; Bio-Rad).

In vitro termination assays

RNAP and ρ for in vitro assays were purified as described before³³. RNAP holoenzymes were assembled by mixing the core RNAP with a three-fold molar excess of σ⁷⁰ transcription initiation factor, followed by incubation at 30 °C for 20 min. DNA templates were generated by PCR amplification with primers listed in Supplementary Table 3 and purified by PCR cleanup kit (Qiagen). Halted A26 ECs were formed at 37 °C for 12 min by mixing 50 nM RNAP holoenzyme with 25 nM DNA template, 100 μM ApU, 10 μM ATP and UTP, 2 μM GTP and 5 Ci/mmol [α³²P] GTP in ρ termination buffer (40 mM Tris-Cl, 50 mM KCl, 5 mM MgCl₂, 0.1 mM DTT, 3% (v/v) glycerol, pH 7.9). After addition of ρ (25 nM) and Rof variants (5 μM), the reactions were incubated for 3 min at 37 °C. Transcription was restarted by addition of a pre-warmed (at 37 °C) mixture containing 150 μM NTPs and 50 μg/ml rifapentin and incubated at 37 °C for 7 min. Reactions were quenched by addition of an equal volume of stop buffer (45 mM Tris-borate, 10 M urea, 20 mM EDTA, 0.2% xylene cyanol, 0.2% bromophenol blue, pH 8.3) and separated by denaturing 5% PAGE (7 M urea, 0.5X TBE). Gels were dried and products were visualized using a Typhoon FLA 9000 PhosphorImaging system (GE Healthcare). Readthrough and termination RNA products were quantitated with ImageQuant and Microsoft Excel software.

Rof and ρ distribution in Pseudomonadota

Rof and ρ distribution (Supplementary Data 1) were estimated using Rof Pfam (PF07073) and ρ TIGR (TIGR00767) models in Annotree⁵⁹ with an e-value 10^-5. The phylogenetic tree was downloaded from Annotree. The result was visualized in R (version 4.2.2) with ggtree package⁶⁰.

Sequence conservation and genomic context analysis

To investigate the conservation of Rof and ρ in Pseudomonadota, the GTDB⁶¹ bacterial taxonomy list (r_202) was downloaded. Only complete genomes were used to build the database. Three representatives from each Genus of Pseudomonadota were selected randomly. Rof was identified with the Pfam model. To identify ρ, Pfam model Rho_RNA_bind (PF07497) was searched against the representative database using hmmsearch (version 3.3)⁶². The identified ρ-like proteins were further confirmed by searching NCBI HMM model (NF006886.1) against them using hmmsearch⁶² (Supplementary Data 1).

To further investigate the conservation of Rof in Enterobacteriaceae and Vibrionaceae, reference genomes of Enterobacteriaceae, Enterobacteriaceae_A and Vibrionaceae were selected from the GTDB bacterial taxonomy list. One representative from each strain was used to build the database. Again, Rof was identified by its Pfam model (Supplementary Data 1).

Before performing multiple sequence alignment, sequence duplicates were removed using cd-hit (version 4.8.1)⁶³. Sequences were aligned using MUSCLE (version 5.1)⁶⁴ with default settings. Sequence logos were generated using WebLogo (version 3.7.8)⁶⁵.

Enterobacteriaceae and Vibrionaceae genomes containing rof and yaeP were analyzed to determine if their ORFs overlap. The overlaps were determined based on chromosomal location, strand and gene distance. We note that instances of non-overlapping genes could be an artifact of low quantality genome assemblies.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

CryoEM reconstructions have been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb) under accession codes EMD-17875 (ρ₆-Rof₅), EMD-17877 (ρ₅-Rof₄), EMD-17874 (ρ₆-ADP-Rof₅), EMD-17876 (ρ₅-ADP-Rof₄) and EMD-17870 (ρ-rut RNA). Structure coordinates have been deposited in the RCSB Protein Data Bank (https://www.rcsb.org) with accession codes 8PTN (ρ₆-Rof₅), 8PTP (ρ₅-Rof₄), 8PTM (ρ₆-ADP-Rof₅), 8PTO (ρ₅-ADP-Rof₄) and 8PTG (ρ-rut RNA). Uncropped gel images and quantifications are provided in a Source Data file. All other data are contained in the manuscript or the Supplementary Information files. Structure coordinates used in this study are available from the RCSB Protein Data Bank [https://www.rcsb.org] under accession codes 1PVO, 1SG5, 3ICE, 5JJI, 6JIE, 6WA8 and 6Z9P. Source data are provided with this paper.

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Acknowledgements

We are very grateful to P. Lydia Freddolino and Natacha Ruiz for many stimulating discussions. This work was supported by the National Institutes of Health (GM067153 to I.A.), the Deutsche Forschungsgemeinschaft (INST 130/1064-1 FUGG to Freie Universität Berlin; GRK 2473 “Bioactive Peptides”, project number 392923329, to M.C.W.; WA 1126/11-1, project number 433623608, to M.C.W.) and the Berlin University Alliance (501_BIS-CryoFac to M.C.W.). The Sanger Sequencing at the OSU Comprehensive Cancer Center is supported in part by NCI P30 CA0168058.

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Open Access funding enabled and organized by Projekt DEAL.

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Daniela Gjorgjevikj
Present address: Department of Medicine, MRC Laboratory of Molecular Biology, University of Cambridge, Cambridge CB2 0QH, United Kingdom

Authors and Affiliations

Laboratory of Structural Biochemistry, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 6, D-14195 Berlin, Germany
Nelly Said, Tarek Hilal, Tim Luca Selinger, Daniela Gjorgjevikj & Markus C. Wahl
Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH, USA
Mark Finazzo, Bing Wang & Irina Artsimovitch
Research Center of Electron Microscopy and Core Facility BioSupraMol, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Fabeckstr. 36a, D-14195 Berlin, Germany
Tarek Hilal
Macromolecular Crystallography, Helmholtz-Zentrum Berlin für Materialien und Energie, Albert-Einstein-Str. 15, D-12489 Berlin, Germany
Markus C. Wahl

Authors

Nelly Said
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Mark Finazzo
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Tarek Hilal
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Bing Wang
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Tim Luca Selinger
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Daniela Gjorgjevikj
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Irina Artsimovitch
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Markus C. Wahl
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Contributions

N.S., B.W., T.L.S., D.G. and I.A. performed preparative and analytical biochemical experiments. M.F. and I.A. conducted in vivo experiments. B.W. carried out bioinformatic analyses. N.S. and T.H. prepared samples for cryoEM. T.H. collected cryoEM data and performed SPA. N.S. built and refined atomic models. All authors contributed to the analysis of data and the interpretation of the results. N.S. and I.A. wrote the manuscript with contributions from all authors. I.A. and M.C.W. coordinated the project.

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Correspondence to Irina Artsimovitch or Markus C. Wahl.

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Nature Communications thanks Max Gottesman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Said, N., Finazzo, M., Hilal, T. et al. Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation. Nat Commun 15, 3186 (2024). https://doi.org/10.1038/s41467-024-47439-6

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Received: 08 September 2023
Accepted: 01 April 2024
Published: 15 April 2024
DOI: https://doi.org/10.1038/s41467-024-47439-6

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