Tarek Hilal

Over the past decade, numerous studies have shown for several adhesion GPCRs (aGPCRs) that signaling activity is regulated by an intramolecular agonist (termed Stachel ) with a conserved amino acid core located proximal to the N terminus of transmembrane helix 1. Here we present cryo-EM structures of the human ADGRG6 (or GPR126) C-terminal fragment bound to heterotrimeric Gs protein in two distinct conformational states — an “ECL2-in” state that sterically and allosterically limits Stachel engagement and a “ Stachel -bound” active state with a core orientation unknown so far — determined to global resolutions of 3.4 Å and 3.8 Å, respectively. In the “ECL2-in” state the central segment of this loop makes a network of hydrogen-bonding and van-der-Waals contacts with the transmembrane domain. Of note, the conserved TM3–ECL2 cysteines do not form the canonical disulfide bridge in the “ECL2-in” conformation. Structure-guided mutagenesis, peptide stimulation and cellular signaling assays map inactivating and constitutively activating variants to spatially distinct ECL2 regions. Together, these data establish ECL2 as an intramolecular, tunable gate that controls internal-agonist engagement and G-protein activation in GPR126, revealing a structural mechanism that can be exploited to bias aGPCR signaling and might support new ideas for rational ligand design.

Spirochaetota is a deeply rooted bacterial phylum of evolutionary and medical importance. We show that the spirochetal RNA polymerase (RNAP) has a diminished ability to melt promoters compared to its Escherichia coli counterpart. This deficiency is compensated by CarD and is not linked to the phylum's natural resistance to the antibiotic rifampicin. CryoEM analysis revealed that the spirochetal RNAP disengages the -35 element during promoter unwinding, which contrasts with other bacterial RNAPs that release this element later during the initial transcription. Our research also details the unusually tight, non-sequence-specific binding of the spirochetal RNAP holoenzyme to DNA, which may be linked to the highly extended structure of the spirochetal nucleoid. We support these observations with a structural analysis of the dimeric holoenzyme non-specifically bound to DNA. Our results highlight the functional diversity of bacterial transcription systems and lay the foundation for understanding transcription regulation in pathogenic spirochetes.

Spirochaetota is a deeply rooted bacterial phylum of evolutionary and medical importance. We show that the spirochetal RNA polymerase (RNAP) has a diminished ability to melt promoters compared to its Escherichia coli counterpart. This deficiency is compensated by CarD and is not linked to the phylum’s natural resistance to the antibiotic rifampicin. CryoEM analysis revealed that the spirochetal RNAP disengages the −35 element during promoter unwinding, which contrasts with other bacterial RNAPs that release this element later during the initial transcription. Our research also details the unusually tight, non-sequence-specific binding of the spirochetal RNAP holoenzyme to DNA, which may be linked to the highly extended structure of the spirochetal nucleoid. We support these observations with a structural analysis of the dimeric holoenzyme non-specifically bound to DNA. Our results highlight the functional diversity of bacterial transcription systems and lay the foundation for understanding transcription regulation in pathogenic spirochetes.

Metazoan histone mRNAs are a unique class of mRNAs that lack the poly(A) tail present in all other eukaryotic transcripts. Instead, they end in a conserved stem-loop (SL) structure, necessitating a decay mechanism that is distinct from deadenylation-initiated degradation. Here, combining structural and functional approaches, we elucidate molecular mechanisms of initiation of histone mRNA decay. At the end of S-phase, the RNA helicase UPF1, the exoribonuclease 3’hExo and stem-loop binding protein SLBP all contribute to histone mRNA degradation, although how they are mechanistically coupled remained unknown. The cryoEM structure of an UPF1:SL RNA complex, presented here, shows that binding of UPF1 partially melts the RNA stem in the absence of ATP, harnessing the free energy derived from RNA-binding to unwind RNA. This melting event primes the SL-RNA for decay by 3’hExo. Using biochemical and cellular analyses, we demonstrate that SLBP directly engages the UPF1 helicase core to attenuate its unwinding activity and prevent premature degradation. Activation of UPF1 at a later stage promotes SL-RNA decay. We provide direct evidence that UPF1, SLBP and 3’hExo form a degradosome-like assembly that functionally couples SL unwinding and degradation, highlighting a dynamic and intricate network of UPF1-centric interactions that orchestrates timely histone mRNA decay.

Bacterial RNA helicase ρ is a genome sentinel that terminates the synthesis of damaged and junk RNAs that are not translated by the ribosome. It is unclear how ρ is regulated during dormancy or stress, when translation is inefficient and RNAs are vulnerable to ρ-mediated release. We use cryogenic electron microscopy, biochemical, and genetic approaches to show that substitutions of residues in the connector between two ρ domains or ADP promote the formation of extended Escherichia coli ρ filaments. By contrast, (p)ppGpp induces the formation of transient ρ dodecamers. Our results demonstrate that ADP and (p)ppGpp nucleotides bound at subunit interfaces inhibit ρ ring closure that underpins the hexamer activation, thus favoring the assembly of inactive higher-order oligomers. Connector substitutions and antibiotics that inhibit RNA and protein syntheses trigger ρ aggregation in the cell. These and other recent data implicate aggregation as a widespread strategy to tune ρ activity.

Many bacteriophages modulate host transcription to favor expression of their own genomes. Phage satellite P4 polarity suppression protein, Psu, a building block of the viral capsid, inhibits hexameric transcription termination factor, ρ, by presently unknown mechanisms. Our cryogenic electron microscopy structures of ρ-Psu complexes show that Psu dimers clamp two inactive, open ρ rings and promote their expansion to higher-oligomeric states. ATPase, nucleotide binding and nucleic acid binding studies revealed that Psu hinders ρ ring closure and traps nucleotides in their binding pockets on ρ. Structure-guided mutagenesis in combination with growth, pull-down, and termination assays further delineated the functional ρ-Psu interfaces in vivo. Bioinformatic analyses revealed that Psu is associated with a wide variety of phage defense systems across Enterobacteriaceae, suggesting that Psu may regulate expression of anti-phage genes. Our findings show that modulation of the ρ oligomeric state via diverse strategies is a pervasive gene regulatory principle in bacteria.

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.

RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an α-helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a β-barrel, which binds the ribosome. Here, we report structures of ops-paused transcription elongation complexes alone and bound to the autoinhibited and activated RfaH, which reveal swiveled, pre-translocated pause states stabilized by an ops hairpin in the non-template DNA. Autoinhibited RfaH binds and twists the ops hairpin, expanding the RNA:DNA hybrid to 11 base pairs and triggering the KOW release. Once activated, RfaH hyper-stabilizes the pause, which thus requires anti-backtracking factors for escape. Our results suggest that the entire RfaH cycle is solely determined by the ops and RfaH sequences and provide insights into mechanisms of recruitment and metamorphosis of NusG homologs across all life.

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. Our electron microscopic 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 confirmed that the observed ρ-Rof interface is required for Rof-mediated inhibition of cell growth and ρ-termination in vitro . Bioinformatic analyses revealed 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 would be lethal.

Bacterial RNA helicase ρ is a genome sentinel that terminates synthesis of damaged and junk RNAs that are not translated by the ribosome. Co-transcriptional RNA surveillance by ρ is essential for quality control of the transcriptome during optimal growth. However, it is unclear how bacteria protect their RNAs from overzealous ρ during dormancy or stress, conditions common in natural habitats. Here we used cryogenic electron microscopy, biochemical, and genetic approaches to show that residue substitutions, ADP, or ppGpp promote hyper-oligomerization of Escherichia coli ρ. Our results demonstrate that nucleotides bound at subunit interfaces control ρ switching from active hexamers to inactive higher-order oligomers and extended filaments. Polymers formed upon exposure to antibiotics or ppGpp disassemble when stress is relieved, thereby directly linking termination activity to cellular physiology. Inactivation of ρ through hyper-oligomerization is a regulatory strategy shared by RNA polymerases, ribosomes, and metabolic enzymes across all life.