Structural basis of transcription arrest by coliphage HK022 nun in an Escherichia coli RNA polymerase elongation complex
- The Rockefeller University, United States
- Public Health Research Institute, United States
- Columbia University Medical Center, United States
Abstract
Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λΔNA.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.
Data availability
-
CryoEM structure of HK022 Nun - E. coli RNA polymerase elongation complexPublicly available at the RCSB Protein Data Bank (accession no: 5UP6).
-
CryoEM structure of HK022 Nun - E. coli RNA polymerase elongation complexPublicly available at the EMBL-EBI Protein Data Bank in Europe (accession no: EMD-8584).
-
CryoEM structure of crosslinked E.coli RNA polymerase elongation complexPublicly available at the RCSB Protein Data Bank (accession no: 5UPA).
-
CryoEM structure of crosslinked E.coli RNA polymerase elongation complexPublicly available at the EMBL-EBI Protein Data Bank in Europe (accession no: EMD-8585).
-
CryoEM structure of E.coli RNA polymerase elongation complexPublicly available at the RCSB Protein Data Bank (accession no: 5UPC).
-
CryoEM structure of E.coli RNA polymerase elongation complexPublicly available at the EMBL-EBI Protein Data Bank in Europe (accession no: EMD-8586).
-
Crystal Structure Analysis of the E.coli holoenzymePublicly available at the RCSB Protein Data Bank (accession no: 4LJZ).
-
Crystal structure of the T. thermophilus RNAP polymerase elongation complex with the NTP substrate analogPublicly available at the RCSB Protein Data Bank (accession no: 2O5J).
Article and author information
Author details
Funding
National Institutes of Health (R35 GM118130)
- Seth A Darst
National Institutes of Health (R01 GM037219)
- Max E Gottesman
National Institutes of Health (P41 GM103314)
- Brian T Chait
Public Health Research Institute Research Support grant
- Arkady Mustaev
National Institutes of Health (P41 GM109824)
- Brian T Chait
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Kang et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,851
- views
-
- 793
- downloads
-
- 118
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Structural basis of transcription arrest by coliphage HK022 nun in an Escherichia coli RNA polymerase elongation complex
eLife 6:e25478.
https://doi.org/10.7554/eLife.25478
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
-
- Biochemistry and Chemical Biology
- Computational and Systems Biology
Protein–protein interactions are fundamental to understanding the molecular functions and regulation of proteins. Despite the availability of extensive databases, many interactions remain uncharacterized due to the labor-intensive nature of experimental validation. In this study, we utilized the AlphaFold2 program to predict interactions among proteins localized in the nuage, a germline-specific non-membrane organelle essential for piRNA biogenesis in Drosophila. We screened 20 nuage proteins for 1:1 interactions and predicted dimer structures. Among these, five represented novel interaction candidates. Three pairs, including Spn-E_Squ, were verified by co-immunoprecipitation. Disruption of the salt bridges at the Spn-E_Squ interface confirmed their functional importance, underscoring the predictive model’s accuracy. We extended our analysis to include interactions between three representative nuage components—Vas, Squ, and Tej—and approximately 430 oogenesis-related proteins. Co-immunoprecipitation verified interactions for three pairs: Mei-W68_Squ, CSN3_Squ, and Pka-C1_Tej. Furthermore, we screened the majority of Drosophila proteins (~12,000) for potential interaction with the Piwi protein, a central player in the piRNA pathway, identifying 164 pairs as potential binding partners. This in silico approach not only efficiently identifies potential interaction partners but also significantly bridges the gap by facilitating the integration of bioinformatics and experimental biology.
