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C Richardson - One of the best experts on this subject based on the ideXlab platform.
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heterohexamer of 56 and 63 kda gene 4 helicase primase of bacterioPhage T7 in dna replication
Journal of Biological Chemistry, 2012Co-Authors: Huidong Zhang, Arkadiusz W Kulczyk, C RichardsonAbstract:BacterioPhage T7 expresses two forms of gene 4 protein (gp4). The 63-kDa full-length gp4 contains both the helicase and primase domains. T7 Phage also express a 56-kDa truncated gp4 lacking the zinc binding domain of the primase; the protein has helicase activity but no DNA-dependent primase activity. Although T7 Phage grow better when both forms are present, the role of the 56-kDa gp4 is unknown. The two molecular weight forms oligomerize by virtue of the helicase domain to form heterohexamers. The 56-kDa gp4 and any mixture of 56- and 63-kDa gp4 show higher helicase activity in DNA unwinding and strand-displacement DNA synthesis than that observed for the 63-kDa gp4. However, single-molecule measurements show that heterohexamers have helicase activity similar to the 63-kDa gp4 hexamers. In oligomerization assays the 56-kDa gp4 and any mixture of the 56- and 63-kDa gp4 oligomerize to form more hexamers than does the 63-kDa gp4. The zinc binding domain of the 63-kDa gp4 interferes with hexamer formation, an inhibition that is relieved by the insertion of the 56-kDa species. Compared with the 63-kDa gp4, heterohexamers synthesize a reduced amount of oligoribonucleotides, mediated predominately by the 63-kDa subunits via a cis mode. During coordinated DNA synthesis 7% of the tetraribonucleotides synthesized are used as primers by both heterohexamers and hexamers of the 63-kDa gp4. Overall, an equimolar mixture of the two forms of gp4 shows the highest rate of DNA synthesis during coordinated DNA synthesis.
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direct role for the rna polymerase domain of T7 primase in primer delivery
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: C RichardsonAbstract:Gene 4 protein (gp4) encoded by bacterioPhage T7 contains a C-terminal helicase and an N-terminal primase domain. After synthesis of tetraribonucleotides, gp4 must transfer them to the polymerase for use as primers to initiate DNA synthesis. In vivo gp4 exists in two molecular weight forms, a 56-kDa form and the full-length 63-kDa form. The 56-kDa gp4 lacks the N-terminal Cys4 zinc-binding motif important in the recognition of primase sites in DNA. The 56-kDa gp4 is defective in primer synthesis but delivers a wider range of primers to initiate DNA synthesis compared to the 63-kDa gp4. Suppressors exist that enable the 56-kDa gp4 to support the growth of T7 Phage lacking gene 4 (T7Δ4). We have identified 56-kDa DNA primases defective in primer delivery by screening for their ability to support growth of T7Δ4 Phage in the presence of this suppressor. Trp69 is critical for primer delivery. Replacement of Trp69 with lysine in either the 56- or 63-kDa gp4 results in defective primer delivery with other functions unaffected. DNA primase harboring lysine at position 69 fails to stabilize the primer on DNA. Thus, a primase subdomain not directly involved in primer synthesis is involved in primer delivery. The stabilization of the primer by DNA primase is necessary for DNA polymerase to initiate synthesis.
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an in trans interaction at the interface of the helicase and primase domains of the hexameric gene 4 protein of bacterioPhage T7 modulates their activities
Journal of Biological Chemistry, 2009Co-Authors: C RichardsonAbstract:Abstract DNA helicase and primase are essential for DNA replication. The helicase unwinds the DNA to provide single-stranded templates for DNA polymerase. The primase catalyzes the synthesis of oligoribonucleotides for the initiation of lagging strand synthesis. The two activities reside in a single polypeptide encoded by gene 4 of bacterioPhage T7. Their coexistence within the same polypeptide facilitates their coordination during DNA replication. One surface of helix E within the helicase domain is positioned to interact with the primase domain and the linker connecting the two domains within the functional hexamer. The interaction occurs in trans such that helix E interacts with the primase domain and the linker of the adjacent subunit. Most alterations of residues on the surface of helix E (Arg404, Lys408, Tyr411, and Gly415) eliminate the ability of the altered proteins to complement growth of T7 Phage lacking gene 4. Both Tyr411 and Gly415 are important in oligomerization of the protein. Alterations G415V and K408A simultaneously influence helicase and primase activities in opposite manners that mimic events observed during coordinated DNA synthesis. The results suggest that Asp263 located in the linker of one subunit can interact with Tyr411, Lys408, or Arg404 in helix E of the adjacent subunit depending on the oligomerization state. Thus the switch in contacts between Asp263 and its three interacting residues in helix E of the adjacent subunit results in conformational changes that modulate helicase and primase activity.
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acidic residues in the nucleotide binding site of the bacterioPhage T7 dna primase
Journal of Biological Chemistry, 2005Co-Authors: C RichardsonAbstract:Abstract DNA primases catalyze the synthesis of oligoribonucleotides to initiate lagging strand DNA synthesis during DNA replication. Like other prokaryotic homologs, the primase domain of the gene 4 helicase-primase of bacterioPhage T7 contains a zinc motif and a catalytic core. Upon recognition of the sequence, 5′-GTC-3′ by the zinc motif, the catalytic site condenses the cognate nucleotides to produce a primer. The TOPRIM domain in the catalytic site contains several charged residues presumably involved in catalysis. Each of eight acidic residues in this region was replaced with alanine, and the properties of the altered primases were examined. Six of the eight residues (Glu-157, Glu-159, Asp-161, Asp-207, Asp-209, and Asp-237) are essential in that altered gene 4 proteins containing these mutations cannot complement T7 Phage lacking gene 4 for T7 growth. These six altered gene 4 proteins can neither synthesize primers de novo nor extend an oligoribonucleotide. Despite the inability to catalyze phosphodiester bond formation, the altered proteins recognize the sequence 5′-GTC-3′ in the template and deliver preformed primer to T7 DNA polymerase. The alterations in the TOPRIM domain result in the loss of binding affinity for ATP as measured by surface plasmon resonance assay together with ATP-agarose affinity chromatography.
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the linker region between the helicase and primase domains of the gene 4 protein of bacterioPhage T7 role in helicase conformation and activity
Journal of Biological Chemistry, 2004Co-Authors: C RichardsonAbstract:Abstract The gene 4 protein of bacterioPhage T7 provides both helicase and primase activities. The C-terminal helicase domain is responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding whereas the N-terminal primase domain is responsible for template-directed oligoribonucleotide synthesis. A 26 amino acid linker region (residues 246-271) connects the two domains and is essential for the formation of functional hexamers. In order to further dissect the role of the linker region, three residues (Ala257, Pro259, and Asp263) that was disordered in the crystal structure of the hexameric helicase fragment were substituted with all amino acids, and the altered proteins were analyzed for their ability to support growth of T7 Phage lacking gene 4. The in vivo screening revealed Ala257 and Asp263 to be essential whereas Pro259 could be replaced with any amino acid without loss of function. Selected gene 4 proteins with substitution for Ala257 or Asp263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize. In the presence of Mg2+, all of the altered proteins oligomerize. However, in the absence of divalent ion, alterations at position 257 increase the extent of oligomerization whereas those at position 263 reduce oligomer formation. Although dTTP hydrolysis activity is reduced only 2-3-fold, none of the altered gene 4 proteins can translocate effectively on single-strand DNA, and they cannot mediate the unwinding of duplex DNA. Primer synthesis catalyzed by the altered proteins is relatively normal on a short DNA template but it is severely impaired on longer templates where translocation is required. The results suggest that the linker region not only connects the two domains of the gene 4 protein and participates in oligomerization, but also contributes to helicase activity by mediating conformations within the functional hexamer.
Fumio Sugawara - One of the best experts on this subject based on the ideXlab platform.
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using the qcm biosensor based T7 Phage display combined with bioinformatics analysis for target identification of bioactive small molecule
Methods of Molecular Biology, 2018Co-Authors: Yoichi Takakusagi, Kaori Takakusagi, Fumio Sugawara, Kengo SakaguchiAbstract:: Identification of target proteins that directly bind to bioactive small molecule is of great interest in terms of clarifying the mode of action of the small molecule as well as elucidating the biological phenomena at the molecular level. Of the experimental technologies available, T7 Phage display allows comprehensive screening of small molecule-recognizing amino acid sequence from the peptide libraries displayed on the T7 Phage capsid. Here, we describe the T7 Phage display strategy that is combined with quartz-crystal microbalance (QCM) biosensor for affinity selection platform and bioinformatics analysis for small molecule-recognizing short peptides. This method dramatically enhances efficacy and throughput of the screening for small molecule-recognizing amino acid sequences without repeated rounds of selection. Subsequent execution of bioinformatics programs allows combinatorial and comprehensive target protein discovery of small molecules with its binding site, regardless of protein sample insolubility, instability, or inaccessibility of the fixed small molecules to internally located binding site on larger target proteins when conventional proteomics approaches are used.
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Identification and characterization of the direct interaction between methotrexate (MTX) and high-mobility group box 1 (HMGB1) protein. PLoS One. 2013;8(5):e63073. • We accept pre-submission inquiries • Our selector tool helps you to find the most relevan
2016Co-Authors: Yuki Kuroiwa, Tomoe Kusayanagi, Yoichi Takakusagi, Kouji Kuramochi, Kengo Sakaguchi, Takahiko Imai, Tomoko Hirayama, Ichiaki Ito, Michiteru Yoshida, Fumio SugawaraAbstract:Background: Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. Methodology/Result: Here, we performed a screening of MTX-binding proteins using T7 Phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 Phage display screen, we identified T7 Phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.5060.03 mM for Al and 0.2460.01 mM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macroPhage-like cells (RAW 264.7), TNF-a release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited i
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ridaifen b a tamoxifen derivative directly binds to grb10 interacting gyf protein 2
Bioorganic & Medicinal Chemistry, 2013Co-Authors: Senko Tsukuda, Tomoe Kusayanagi, Shinji Kamisuki, Yoichi Takakusagi, Toshifumi Takeuchi, Eri Umeda, Chihiro Watanabe, Yuta Tosaki, Isamu Shiina, Fumio SugawaraAbstract:Ridaifen B (RID-B) is a tamoxifen derivative that potently inhibits breast tumor growth. RID-B was reported to show anti-proliferating activity for a variety of estrogen receptor (ER)-positive human cancer cells. Interestingly, RID-B was also reported to possess higher potency than that of tamoxifen even for some ER-negative cells, suggesting an ER-independent mechanism of action. In this study, a T7 Phage display screen and subsequent binding analyses have identified Grb10 interacting GYF protein 2 (GIGYF2) as a RID-B-binding protein. Using a cell-based assay, the Akt phosphorylation level mediated by GIGYF2 was found to have decreased in the presence of RID-B.
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Identification and Characterization of the Direct Interaction between Methotrexate (MTX) and High-Mobility Group Box 1 (HMGB1) Protein
2013Co-Authors: Yuki Kuroiwa, Tomoe Kusayanagi, Yoichi Takakusagi, Kouji Kuramochi, Kengo Sakaguchi, Takahiko Imai, Tomoko Hirayama, Ichiaki Ito, Michiteru Yoshida, Fumio SugawaraAbstract:BackgroundMethotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood.Methodology/ResultHere, we performed a screening of MTX-binding proteins using T7 Phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 Phage display screen, we identified T7 Phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.50±0.03 µM for Al and 0.24±0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macroPhage-like cells (RAW 264.7), TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX.Conclusions/SignificanceThese data demonstrate that HMGB1 is a direct binding protein of MTX. Moreover, binding of MTX to RAGE-binding region in HMGB1 inhibited the HMGB1/RAGE interaction at the molecular and cellular levels. These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.
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synthesis of a biotinylated camptothecin derivative and determination of the binding sequence by T7 Phage display technology
Bioorganic & Medicinal Chemistry Letters, 2005Co-Authors: Yoichi Takakusagi, Kouji Kuramochi, Kengo Sakaguchi, Keisuke Ohta, Kengo Morohashi, Susumu Kobayashi, Fumio SugawaraAbstract:A biotinylated derivative of the anti-tumor agent camptothecin (CPT) was synthesized and used in a Phage display assay to identify drug-binding sequences. After three rounds of selection using C20-biotinylated CPT (CPT-20-B) as bait, a CPT-20-B-binding sequence, NSSQSARR, was identified.
Yoichi Takakusagi - One of the best experts on this subject based on the ideXlab platform.
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biosensor based high throughput biopanning and bioinformatics analysis strategy for the global validation of drug protein interactions
Journal of Visualized Experiments, 2020Co-Authors: Yoichi TakakusagiAbstract:Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 Phage-displayed short peptides that can be easily displayed on the Phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as "broken relics", of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.
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using the qcm biosensor based T7 Phage display combined with bioinformatics analysis for target identification of bioactive small molecule
Methods of Molecular Biology, 2018Co-Authors: Yoichi Takakusagi, Kaori Takakusagi, Fumio Sugawara, Kengo SakaguchiAbstract:: Identification of target proteins that directly bind to bioactive small molecule is of great interest in terms of clarifying the mode of action of the small molecule as well as elucidating the biological phenomena at the molecular level. Of the experimental technologies available, T7 Phage display allows comprehensive screening of small molecule-recognizing amino acid sequence from the peptide libraries displayed on the T7 Phage capsid. Here, we describe the T7 Phage display strategy that is combined with quartz-crystal microbalance (QCM) biosensor for affinity selection platform and bioinformatics analysis for small molecule-recognizing short peptides. This method dramatically enhances efficacy and throughput of the screening for small molecule-recognizing amino acid sequences without repeated rounds of selection. Subsequent execution of bioinformatics programs allows combinatorial and comprehensive target protein discovery of small molecules with its binding site, regardless of protein sample insolubility, instability, or inaccessibility of the fixed small molecules to internally located binding site on larger target proteins when conventional proteomics approaches are used.
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Identification and characterization of the direct interaction between methotrexate (MTX) and high-mobility group box 1 (HMGB1) protein. PLoS One. 2013;8(5):e63073. • We accept pre-submission inquiries • Our selector tool helps you to find the most relevan
2016Co-Authors: Yuki Kuroiwa, Tomoe Kusayanagi, Yoichi Takakusagi, Kouji Kuramochi, Kengo Sakaguchi, Takahiko Imai, Tomoko Hirayama, Ichiaki Ito, Michiteru Yoshida, Fumio SugawaraAbstract:Background: Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. Methodology/Result: Here, we performed a screening of MTX-binding proteins using T7 Phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 Phage display screen, we identified T7 Phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.5060.03 mM for Al and 0.2460.01 mM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macroPhage-like cells (RAW 264.7), TNF-a release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited i
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multimodal biopanning of T7 Phage displayed peptides reveals angiomotin as a potential receptor of the anti angiogenic macrolide roxithromycin
European Journal of Medicinal Chemistry, 2015Co-Authors: Kaori Takakusagi, Yoichi Takakusagi, Takahiro Suzuki, Aya Toizaki, Aiko Suzuki, Yaichi Kawakatsu, Madoka Watanabe, Yukihiro Saito, Ryushi Fukuda, Atsuo NakazakiAbstract:Roxithromycin (RXM) is a semi-synthetic fourteen-membered macrolide antibiotic that shows anti-angiogenic activity in solid tumors. In the present study, we conducted biopanning of T7 Phage-displayed peptides either on a 96-well formatted microplate, a flow injection-type quartz-crystal microbalance (QCM) biosensor, or a cuvette-type QCM. RXM-selected peptides of different sequence, length and number were obtained from each mode of screening. Subsequent bioinformatics analysis of the RXM-selected peptides consistently gave positive scores for the extracellular domain (E458-T596) of angiomotin (Amot), indicating that this may comprise a binding region for RXM. Bead pull down assay and QCM analysis confirmed that RXM directly interacts with Amot via the screen-guided region, which also corresponds to the binding site for the endogenous anti-angiogenic inhibitor angiostatin (Anst). Thus, multimodal biopanning of T7PD revealed that RXM binds to the extracellular domain on Amot as a common binding site with Anst, leading to inhibition of angiogenesis-dependent tumor growth and metastasis. These data might explain the molecular basis underlying the mechanism of action for the anti-angiogenic activity of RXM.
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ridaifen b a tamoxifen derivative directly binds to grb10 interacting gyf protein 2
Bioorganic & Medicinal Chemistry, 2013Co-Authors: Senko Tsukuda, Tomoe Kusayanagi, Shinji Kamisuki, Yoichi Takakusagi, Toshifumi Takeuchi, Eri Umeda, Chihiro Watanabe, Yuta Tosaki, Isamu Shiina, Fumio SugawaraAbstract:Ridaifen B (RID-B) is a tamoxifen derivative that potently inhibits breast tumor growth. RID-B was reported to show anti-proliferating activity for a variety of estrogen receptor (ER)-positive human cancer cells. Interestingly, RID-B was also reported to possess higher potency than that of tamoxifen even for some ER-negative cells, suggesting an ER-independent mechanism of action. In this study, a T7 Phage display screen and subsequent binding analyses have identified Grb10 interacting GYF protein 2 (GIGYF2) as a RID-B-binding protein. Using a cell-based assay, the Akt phosphorylation level mediated by GIGYF2 was found to have decreased in the presence of RID-B.
Sivaramesh Wigneshweraraj - One of the best experts on this subject based on the ideXlab platform.
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the rna binding protein hfq assembles into foci like structures in nitrogen starved escherichia coli
Journal of Biological Chemistry, 2020Co-Authors: Josh Mcquail, Lynn Burchell, Amy Switzer, Sivaramesh WigneshwerarajAbstract:The initial adaptive responses to nutrient depletion in bacteria often occur at the level of gene expression. Hfq is an RNA-binding protein present in diverse bacterial lineages that contributes to many different aspects of RNA metabolism during gene expression. Using photoactivated localization microscopy and single-molecule tracking, we demonstrate that Hfq forms a distinct and reversible focus-like structure in Escherichia coli specifically experiencing long-term nitrogen starvation. Using the ability of T7 Phage to replicate in nitrogen-starved bacteria as a biological probe of E. coli cell function during nitrogen starvation, we demonstrate that Hfq foci have a role in the adaptive response of E. coli to long-term nitrogen starvation. We further show that Hfq foci formation does not depend on gene expression once nitrogen starvation has set in and occurs indepen-dently of the transcription factor N-regulatory protein C, which activates the initial adaptive response to N starvation in E. coli These results serve as a paradigm to demonstrate that bacterial adaptation to long-term nutrient starvation can be spatiotemporally coordinated and can occur independently of de novo gene expression during starvation.
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T7 Phage factor required for managing rpos inescherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Aline Tabibsalazar, Udi Qimron, Bing Liu, Lynn Burchell, Declan Barker, Steve Matthews, Sivaramesh WigneshwerarajAbstract:T7 development in Escherichia coli requires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. Although 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 growing E. coli as a consequence of the buildup of guanosine pentaphosphate [(p)ppGpp] during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development in E. coli cells 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.
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substitutions in the escherichia coli rna polymerase inhibitor T7 gp2 that allow inhibition of transcription when the primary interaction interface between gp2 and rna polymerase becomes compromised
Microbiology, 2012Co-Authors: Andrey Shadrin, Konstantin Severinov, Steve Matthews, Carol Sheppard, Sivaramesh WigneshwerarajAbstract:The Escherichia coli-infecting bacterioPhage T7 encodes a 7 kDa protein, called Gp2, which is a potent inhibitor of the host RNA polymerase (RNAp). Gp2 is essential for T7 Phage development. The interaction site for Gp2 on the E. coli RNAp is the β′ jaw domain, which is part of the DNA binding channel. The binding of Gp2 to the β′ jaw antagonizes several steps associated with interactions between the RNAp and promoter DNA, leading to inhibition of transcription at the open promoter complex formation step. In the structure of the complex formed between Gp2 and a fragment of the β′ jaw, amino acid residues in the β3 strand of Gp2 contribute to the primary interaction interface with the β′ jaw. The 7009 E. coli strain is resistant to T7 because it carries a charge reversal point mutation in the β′ jaw that prevents Gp2 binding. However, a T7 Phage encoding a mutant form of Gp2, called Gp2β, which carries triple amino acid substitutions E24K, F27Y and R56C, can productively infect this strain. By studying the molecular basis of inhibition of RNAp from the 7009 strain by Gp2β, we provide several lines of evidence that the E24K and F27Y substitutions facilitate an interaction with RNAp when the primary interaction interface with the β′ jaw is compromised. The proposed additional interaction interface between RNAp and Gp2 may contribute to the multipronged mechanism of transcription inhibition by Gp2.
Udi Qimron - One of the best experts on this subject based on the ideXlab platform.
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T7 Phage factor required for managing rpos inescherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Aline Tabibsalazar, Udi Qimron, Bing Liu, Lynn Burchell, Declan Barker, Steve Matthews, Sivaramesh WigneshwerarajAbstract:T7 development in Escherichia coli requires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. Although 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 growing E. coli as a consequence of the buildup of guanosine pentaphosphate [(p)ppGpp] during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development in E. coli cells 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.
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a T7 Phage factor required for managing rpos in escherichia coli
bioRxiv, 2018Co-Authors: Aline Tabibsalazar, Udi Qimron, Bing Liu, Lynn Burchell, Steve Matthews, Declan Barkar, Ramesh WigneshwerarajAbstract:T7 development in Escherichia coli requires 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 growing E. coli as a consequence of buildup of guanosine pentaphosphate ((p)ppGpp) during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development in E. coli cells 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.
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full shut off of escherichia coli rna polymerase by T7 Phage requires a small Phage encoded dna binding protein
Nucleic Acids Research, 2017Co-Authors: Aline Tabibsalazar, Moran G. Goren, Ido Yosef, Udi Qimron, Bing Liu, Andrey Shadrin, Lynn Burchell, Zhexin Wang, Zhihao Wang, Konstantin SeverinovAbstract:Infection of Escherichia coli by the T7 Phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the Phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how Phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient Phage progeny development.
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Extending the Host Range of BacterioPhage Particles for DNA Transduction
Molecular Cell, 2017Co-Authors: Ido Yosef, Moran G. Goren, Rea Globus, Shahar Molshanski-mor, Udi QimronAbstract:A major limitation in using bacterioPhage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic Phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into Phage-restrictive hosts. To extend the host range of T7 Phage for DNA transduction, we have designed hybrid particles displaying various Phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 Phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel Phage-restrictive hosts, providing a platform for myriad applications that require this ability.
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efficient engineering of a bacterioPhage genome using the type i e crispr cas system
RNA Biology, 2014Co-Authors: Ruth Kiro, Dror Shitrit, Udi QimronAbstract:The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system has recently been used to engineer genomes of various organisms, but surprisingly, not those of bacterioPhages (Phages). Here we present a method to genetically engineer the Escherichia coli Phage T7 using the type I-E CRISPR-Cas system. T7 Phage genome is edited by homologous recombination with a DNA sequence flanked by sequences homologous to the desired location. Non-edited genomes are targeted by the CRISPR-Cas system, thus enabling isolation of the desired recombinant Phages. This method broadens CRISPR Cas-based editing to Phages and uses a CRISPR-Cas type other than type II. The method may be adjusted to genetically engineer any bacterioPhage genome.
