A Bacteriophage DNA Mimic Protein Employs a Non-specific Strategy to Inhibit the Bacterial RNA Polymerase

DNA mimicry by proteins is a strategy that employed by some proteins to occupy the binding sites of the DNA-binding proteins and deny further access to these sites by DNA. Such proteins have been found in bacteriophage, eukaryotic virus, prokaryotic, and eukaryotic cells to imitate non-coding functions of DNA. Here, we report another phage protein Gp44 from bacteriophage SPO1 of Bacillus subtilis, employing mimicry as part of unusual strategy to inhibit host RNA polymerase. Consisting of three simple domains, Gp44 contains a DNA binding motif, a flexible DNA mimic domain and a random-coiled domain. Gp44 is able to anchor to host genome and interact bacterial RNA polymerase via the β and β′ subunit, resulting in bacterial growth inhibition. Our findings represent a non-specific strategy that SPO1 phage uses to target different bacterial transcription machinery regardless of the structural variations of RNA polymerases. This feature may have potential applications like generation of genetic engineered phages with Gp44 gene incorporated used in phage therapy to target a range of bacterial hosts.

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Ureidothiophene inhibits interaction of bacterial RNA polymerase with –10 promotor element

Nucleic Acids Research ◽

10.1093/nar/gkaa591 ◽

2020 ◽

Vol 48 (14) ◽

pp. 7914-7923

Author(s):

John Harbottle ◽

Nikolay Zenkin

Keyword(s):

Complex Formation ◽

Rna Polymerase ◽

Conformational Changes ◽

Early Stage ◽

Chemical Compounds ◽

Initiation Factor ◽

Promoter Element ◽

Bacterial Transcription ◽

Bacterial Rna ◽

Novel Target

Abstract Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter –10 element, while not affecting interactions with –35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of –10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with –10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.

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The Phage-Encoded N-Acetyltransferase Rac Mediates Inactivation of Pseudomonas aeruginosa Transcription by Cleavage of the RNA Polymerase Alpha Subunit

Viruses ◽

10.3390/v12090976 ◽

2020 ◽

Vol 12 (9) ◽

pp. 976 ◽

Cited By ~ 1

Author(s):

Pieter-Jan Ceyssens ◽

Jeroen De Smet ◽

Jeroen Wagemans ◽

Natalia Akulenko ◽

Evgeny Klimuk ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Rna Polymerase ◽

Transcription Initiation ◽

Alpha Subunit ◽

Regulation Mechanism ◽

Post Translational Modification ◽

Α Subunit ◽

Bacterial Transcription ◽

Highly Active ◽

Bacterial Rna

In this study, we describe the biological function of the phage-encoded protein RNA polymerase alpha subunit cleavage protein (Rac), a predicted Gcn5-related acetyltransferase encoded by phiKMV-like viruses. These phages encode a single-subunit RNA polymerase for transcription of their late (structure- and lysis-associated) genes, whereas the bacterial RNA polymerase is used at the earlier stages of infection. Rac mediates the inactivation of bacterial transcription by introducing a specific cleavage in the α subunit of the bacterial RNA polymerase. This cleavage occurs within the flexible linker sequence and disconnects the C-terminal domain, required for transcription initiation from most highly active cellular promoters. To achieve this, Rac likely taps into a novel post-translational modification (PTM) mechanism within the host Pseudomonas aeruginosa. From an evolutionary perspective, this novel phage-encoded regulation mechanism confirms the importance of PTMs in the prokaryotic metabolism and represents a new way by which phages can hijack the bacterial host metabolism.

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A T7 phage factor required for managing RpoS inEscherichia coli

10.1101/262279 ◽

2018 ◽

Author(s):

Aline Tabib-Salazar ◽

Bing Liu ◽

Declan Barker ◽

Lynn Burchell ◽

Udi Qimron ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Stationary Phase ◽

Stationary Phases ◽

Specific Inhibitor ◽

E Coli ◽

Bacterial Transcription ◽

Bacterial Rna ◽

Phage T7 ◽

T7 Phage

T7 development inEscherichia colirequires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. While the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit EσS, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growingE. colias a consequence of buildup of guanosine pentaphosphate ((p)ppGpp) during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development inE. colicells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.Significance statementVirus that infect bacteria (phages) represent the most abundant living entities on the planet and many aspects of our fundamental knowledge of phage-bacteria relationships have been derived in the context of exponentially growing bacteria. In the case of the prototypicalEscherichia coliphage T7, specific inhibition of the housekeeping form of the RNA polymerase (Eσ70) by a T7 protein, called Gp2, is essential for the development of viral progeny. We now reveal that T7 uses a second specific inhibitor that selectively inhibits the stationary phase RNAP (EσS), which enables T7 to develop well in exponentially growing and stationary phase bacteria. The results have broad implications for our understanding of phage-bacteria relationships and therapeutic application of phages.

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