scholarly journals Structural basis of transcription arrest by coliphage HK022 Nun in an Escherichia coli RNA polymerase elongation complex

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jin Young Kang ◽  
Paul Dominic B Olinares ◽  
James Chen ◽  
Elizabeth A Campbell ◽  
Arkady Mustaev ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Terminal Segment ◽  
Structural Basis ◽  
Elongation Complex ◽  
Structural Framework ◽  
Cryo Electron Microscopy ◽  
Active Site Cleft

Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures of Escherichia coli RNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP β and β’ subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs.

scholarly journals Structural basis for transcription activation by Crl through tethering of σS and RNA polymerase

2019 ◽  
Author(s):  
Alexis Jaramillo Cartagena ◽  
Amy B. Banta ◽  
Nikhil Sathyan ◽  
Wilma Ross ◽  
Richard L. Gourse ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Structural Basis ◽  
Structural Element ◽  
Cryo Electron Microscopy ◽  
Promoter Dna ◽  
Transcription Activators

AbstractIn bacteria, a primary σ factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ factors are negatively regulated by anti-σ factors. In Escherichia coli, Salmonella enterica, and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σS regulon by promoting the association of σS with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σS-RNAP in an open promoter complex with a σS regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σS (σS), the structure, along with p-benzoylphenylalanine crosslinking, reveals that Crl interacts with a structural element of the RNAP β’ subunit we call the β’-clamp-toe (β’CT). Deletion of the β’CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β’CT interaction. We conclude that Crl activates σS-dependent transcription in part through stabilizing σS-RNAP by tethering σS and the β’CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.Significance StatementIn bacteria, multiple σ factors can bind to a common core RNA polymerase (RNAP) to alter global transcriptional programs in response to environmental stresses. Many γ-proteobacteria, including the pathogens Yersinia pestis, Vibrio cholera, Escherichia coli, and Salmonella typhimurium, encode Crl, a transcription factor that activates σS-dependent genes. Many of these genes are involved in processes important for infection, such as biofilm formation. We determined a high-resolution cryo-electron microscopy structure of a Crl-σS-RNAP transcription initiation complex. The structure, combined with biochemical experiments, shows that Crl stabilizes σS-RNAP by tethering σS directly to the RNAP.

scholarly journals Structural basis of transcription-translation coupling

Science ◽  
2020 ◽  
Vol 369 (6509) ◽  
pp. 1359-1365 ◽  
Author(s):  
Chengyuan Wang ◽  
Vadim Molodtsov ◽  
Emre Firlar ◽  
Jason T. Kaelber ◽  
Gregor Blaha ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Transcription Factors ◽  
Rna Polymerase ◽  
Active Center ◽  
Messenger Rna ◽  
Coupled Processes ◽  
Structural Basis ◽  
Cryo Electron Microscopy ◽  
Translation Coupling

In bacteria, transcription and translation are coupled processes in which the movement of RNA polymerase (RNAP)–synthesizing messenger RNA (mRNA) is coordinated with the movement of the first ribosome-translating mRNA. Coupling is modulated by the transcription factors NusG (which is thought to bridge RNAP and the ribosome) and NusA. Here, we report cryo–electron microscopy structures of Escherichia coli transcription-translation complexes (TTCs) containing different-length mRNA spacers between RNAP and the ribosome active-center P site. Structures of TTCs containing short spacers show a state incompatible with NusG bridging and NusA binding (TTC-A, previously termed “expressome”). Structures of TTCs containing longer spacers reveal a new state compatible with NusG bridging and NusA binding (TTC-B) and reveal how NusG bridges and NusA binds. We propose that TTC-B mediates NusG- and NusA-dependent transcription-translation coupling.

scholarly journals Author response: Structural basis of transcription arrest by coliphage HK022 Nun in an Escherichia coli RNA polymerase elongation complex

2017 ◽  
Author(s):  
Jin Young Kang ◽  
Paul Dominic B Olinares ◽  
James Chen ◽  
Elizabeth A Campbell ◽  
Arkady Mustaev ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Author Response ◽  
Structural Basis ◽  
Elongation Complex

scholarly journals Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomáš Kouba ◽  
Tomáš Koval’ ◽  
Petra Sudzinová ◽  
Jiří Pospíšil ◽  
Barbora Brezovská ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Rna Synthesis ◽  
Environmental Changes ◽  
Transcription Elongation ◽  
Specific Interactions ◽  
Inactive State ◽  
Transcription Machinery

AbstractRNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.

Interaction of Escherichia coli RNA polymerase with DNA in an elongation complex arrested at a specific psoralen crosslink site

1988 ◽  
Vol 199 (2) ◽  
pp. 277-293 ◽  
Author(s):  
Yun-bo Shi ◽  
Howard Gamper ◽  
Bennett Van Houten ◽  
John E. Hearst
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Elongation Complex

scholarly journals The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hao-Hong Pei ◽  
Tarek Hilal ◽  
Zhuo A. Chen ◽  
Yong-Heng Huang ◽  
Yuan Gao ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Genome Stability ◽  
Slow Growth ◽  
Coiled Coil ◽  
Main Channel ◽  
Dependent Manner ◽  
Secondary Channel

AbstractCellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the β and β′ subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD)2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.

scholarly journals Direct binding of TFEα opens DNA binding cleft of RNA polymerase

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sung-Hoon Jun ◽  
Jaekyung Hyun ◽  
Jeong Seok Cha ◽  
Hoyoung Kim ◽  
Michael S. Bartlett ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Structural Basis ◽  
Transcription Bubble ◽  
Cryo Electron Microscopy ◽  
Direct Binding ◽  
Binding Cleft ◽  
Promoter Dna ◽  
Winged Helix Domain

AbstractOpening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.

scholarly journals Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol

Science ◽  
2020 ◽  
Vol 368 (6496) ◽  
pp. 1211-1219 ◽  
Author(s):  
Lu Zhang ◽  
Yao Zhao ◽  
Yan Gao ◽  
Lijie Wu ◽  
Ruogu Gao ◽  
...  
Keyword(s):  
Cell Wall ◽  
Active Site ◽  
Structural Basis ◽  
Drug Resistant ◽  
Cell Wall Synthesis ◽  
X Ray ◽  
Cryo Electron Microscopy ◽  
Donor And Acceptor ◽  
Biochemical Function ◽  
Glycosyl Donor

The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, we determined cryo–electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.

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