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Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription–translation-coupling quality control in Escherichia coli 1 – 4 . Here we report the preparation of complexes that are functional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopy structures of the complexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer; have RNA threaded through the central channel of Rho; have 60 nucleotides of RNA interacting sequence-specifically with the exterior of Rho and 6 nucleotides of RNA interacting sequence-specifically with the central channel of Rho; have Rho oriented relative to RNAP such that ATP-dependent translocation by Rho exerts mechanical force on RNAP; and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho’s function. Structures presented in this study confirm decades of genetic and biochemical evidence for the mechanism of Rho-dependent termination in bacteria.

All organisms—bacteria, archaea, and eukaryotes—have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription initiation-factor structural module preorganizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate and define details of stepwise, RNA-extension-driven displacement of the “σ-finger” of the bacterial transcription initiation factor σ. The structures reveal that—for both the primary σ-factor and extracytoplasmic (ECF) σ-factors, and for both 5′-triphosphate RNA and 5′-hydroxy RNA—the “σ-finger” is displaced in stepwise fashion, progressively folding back upon itself, driven by collision with the RNA 5′-end, upon extension of nascent RNA from ∼5 nt to ∼10 nt.

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. Here, the authors report cryo-electron microscopy structures of Escherichia coli transcription–translation complexes containing the transcription–translation coupling factor RfaH, showing that RfaH physically bridges RNA polymerase and the ribosome.

The ribonuclease FttA (also known as aCPSF and aCPSF1) mediates factor-dependent transcription termination in archaea 1 – 3 . Here we report the structure of a Thermococcus kodakarensis transcription pre-termination complex comprising FttA, Spt4, Spt5 and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic centre and that enables endonucleolytic cleavage of RNA by FttA, followed by 5′→3′ exonucleolytic cleavage of RNA by FttA and concomitant 5′→3′ translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination. The structure further reveals that Spt5 bridges FttA and the TEC, explaining how Spt5 stimulates FttA-dependent termination. The results reveal functional analogy between bacterial and archaeal factor-dependent termination, functional homology between archaeal and eukaryotic factor-dependent termination, and fundamental mechanistic similarities in factor-dependent termination in bacteria, archaea, and eukaryotes. Cryo-electron microscopy structures of the Thermococcus kodakarensis transcription pre-termination complex suggest a mechanism by which the archaeal termination factor FttA applies mechanical force to a transcription elongation complex to trigger termination, and reveal similarities in factor-dependent termination in bacteria, archaea, and eukaryotes.

In σ-dependent transcriptional pausing, the transcription initiation factor σ, translocating with RNA polymerase (RNAP), makes sequence-specific protein–DNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in σ-dependent pausing, the RNAP active center can access off-pathway “backtracked” states that are substrates for the transcript-cleavage factors of the Gre family and on-pathway “scrunched” states that mediate pause escape. Here, using site-specific protein–DNA photocrosslinking to define positions of the RNAP trailing and leading edges and of σ relative to DNA at the λPR’ promoter, we show directly that σ-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 2–4 bp, and σ-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2–3 bp. Analogous experiments with a library of 4⁷ (∼16,000) transcribed-region sequences show that the state scrunched by 2–3 bp—and only that state—is associated with the consensus sequence, T−3N−2Y−1G+1, (where −1 corresponds to the position of the RNA 3’ end), which is identical to the consensus for pausing in initial transcription and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T−3 resides in the DNA nontemplate strand. A cryoelectron microscopy structure of a complex engaged in σ-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T−3 of the consensus sequence exerts its effects by facilitating scrunching.

Reiterative transcription is a noncanonical form of RNA synthesis in which a nucleotide specified by a single base in the DNA template is repetitively added to the nascent transcript. Here we determined the crystal structure of an RNA polymerase, the bacterial enzyme from Thermus thermophilus, engaged in reiterative transcription during transcription initiation at a promoter resembling the pyrG promoter of Bacillus subtilis. The structure reveals that the reiterative transcript detours from the dedicated RNA exit channel and extends toward the main channel of the enzyme, thereby allowing RNA extension without displacement of the promoter recognition σ-factor. Nascent transcripts containing reiteratively added G residues are eventually extended by nonreiterative transcription, revealing an atypical pathway for the formation of a transcription elongation complex.

Rho and NusG mediate factor-dependent transcription termination in Escherichia coli. Here, we report preparation of complexes functional in factor-dependent termination from RNA polymerase (RNAP), Rho, NusG, and synthetic nucleic-acid scaffolds, and we report cryo-EM structures of complexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer, have RNA threaded through the central channel of Rho, have 60 nt of RNA interacting sequence-specifically with the exterior of Rho and 6 nt of RNA interacting sequence-specifically with the central channel of Rho, have Rho oriented relative to RNAP such that ATP-hydrolysis-dependent translocation by Rho exerts mechanical force on RNAP, and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho function. Cryo-EM reveals the structure of the functional Rho pre-termination complex

Structures recently have been reported of molecular assemblies that mediate transcription-translation coupling in . In these molecular assemblies, termed \"coupled transcription-translation complexes\" or \"TTC-B\", RNA polymerase (RNAP) directly interacts with the ribosome, the transcription elongation factor NusG or its paralog RfaH forms a bridge between RNAP and ribosome, and the transcription elongation factor NusA optionally forms a second bridge between RNAP and ribosome. Here, we report structures of coupled transcription-translation complexes having mRNA spacers between RNAP and ribosome longer than the maximum-length mRNA spacer compatible with formation of TTC-B. The results define a new class of coupled transcription-translation complex, termed \"TTC-LC,\" where \"LC\" denotes \"long-range coupling.\" TTC-LC differs from TTC-B by a ∼60° rotation and ∼70 Å translation of RNAP relative to ribosome, resulting in loss of direct interactions between RNAP and ribosome and creation of a ∼70 Å gap between RNAP and ribosome. TTC-LC accommodates long mRNA spacers by looping out mRNA from the gap between RNAP and ribosome. We present evidence that TTC-LC is an intermediate in assembling and disassembling TTC-B, mediating pre-TTC-B transcription-translation coupling before a ribosome catches up to RNAP, and mediating post-TTC-B transcription-translation coupling after a ribosome stops moving and RNAP continues moving. We show that TTC-B, but not TTC-LC, is severely defective in intrinsic, RNA-hairpin-dependent termination, and that both TTC-B and TTC-LC are severely defective in Rho-dependent termination.

The NusG paralog RfaH mediates bacterial transcription-translation coupling on genes that contain a DNA sequence element, termed an site, required for pausing RNA polymerase (RNAP) and for loading RfaH onto the paused RNAP. Here we report cryo-EM structures of transcription-translation complexes (TTCs) containing RfaH. The results show that RfaH bridges RNAP and the ribosome, with the RfaH N-terminal domain interacting with RNAP, and with 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, due to the more restricted flexibility of the RfaH interdomain linker. The results further show that the structural organization of RfaH-coupled TTCs in the \"loading state,\" in which RNAP and RfaH are located at the 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 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. Cryo-EM reveals the structural basis of transcription-translation coupling by RfaH.

The ribonuclease FttA mediates factor-dependent transcription termination in archaea . Here, we report the structure of a transcription pre-termination complex comprising FttA, Spt4, Spt5, and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic center and that enables endonucleolytic cleavage of RNA by FttA, followed by 5'→3' exonucleolytic cleavage of RNA by FttA and concomitant 5'→3' translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination. The structure further reveals that Spt5 bridges FttA and the TEC, explaining how Spt5 stimulates FttA-dependent termination. The results reveal functional analogy between bacterial and archaeal factor-dependent termination, reveal functional homology between archaeal and eukaryotic factor-dependent termination, and reveal fundamental mechanistic similarities in factor-dependent termination in the three domains of life: bacterial, archaeal, and eukaryotic. Cryo-EM reveals the structure of the archaeal FttA pre-termination complex.