Journal: PLoS Neglected Tropical Diseases

Article Title: Wolbachia Transcription Elongation Factor “Wol GreA” Interacts with α2ββ′σ Subunits of RNA Polymerase through Its Dimeric C-Terminal Domain

doi: 10.1371/journal.pntd.0002930

Figure Lengend Snippet: (A) Purification of Wol GreA from Rosetta strain of E. coli by affinity chromatography. Lane M, protein marker; Lane 1, soluble E. coli proteins following induction with 0.5-mM IPTG at 25°C for 6 h; Lane 2, flow through; Lane 3, washed fraction; Lanes 4-5, eluted fractions of His-tagged purified recombinant Wol GreA at 250-mM imidazole conc. (B) Purification of Wol NTD from E. coli expression host, Rosetta by Ni-NTA column. Lane M, protein marker; Lane 1, soluble E. coli proteins induced with 0.5-mM IPTG at 25°C for 6 h; Lane 2, flow through; Lane 3, washed fraction; Lanes 4–5, elution of recombinant Wol NTD at 250-mM conc. of imidazole. (C) Wol CTD purified from Rosetta strain of E. coli . Lane M, protein marker; Lane 1, soluble E. coli proteins induced under similar conditions as mentioned for Wol GreA; Lane 2, flow through; Lane 3, washed fraction; Lanes 4–5, eluted fractions of purified recombinant Wol CTD. (D) Immune-localization of recombinant Wol GreA and its domains by anti-His monoclonal antibody in Western blot. Lane M, protein marker; Lane 1, ∼18.7-kDa Wol GreA; Lane 2, ∼9-kDa Wol NTD; Lane 3, ∼10.3-kDa Wol CTD.

Article Snippet: The binding of Wol GreA and its domains with E. coli RNAP holoenzyme (Biotech Desk, India) was carried out as described earlier .

Techniques: Purification, Affinity Chromatography, Marker, Recombinant, Expressing, Western Blot

Journal: PLoS Neglected Tropical Diseases

Article Title: Wolbachia Transcription Elongation Factor “Wol GreA” Interacts with α2ββ′σ Subunits of RNA Polymerase through Its Dimeric C-Terminal Domain

doi: 10.1371/journal.pntd.0002930

Figure Lengend Snippet: Using ClustalW, a.a sequence of Wol GreA (wBm, reference sequence: YP_198144.1) on alignment with an array of gram-negative proteobacterial species revealed varying degrees of homology. Wol GreA revealed 90% alignment score with GreA of Wolbachia of Onchocerca ochengi (wOo, reference sequence: YP_006556205.1), 89% with Wolbachia of Drosophila melanogaster (wDm, reference sequence: NP_966418.1), 86% with Wolbachia of Culex quinquefasciatus (wCq, reference sequence: YP_001975932.1), 57% with α-proteobacteria Ehrlichia canis (E. can, reference sequence:YP_302733.1), 47% with Rhizobium leguminosarum (R. leg, reference sequence: YP_002976774.1), 46% with Agrobacterium tumefaciens (A. tum, reference sequence: ZP_12909003.1), 45% with rod-shaped Methylobacterium radiotolerans (M. rad, reference sequence: YP_001753260.1) and 44% with Rickettsia africae (R. afr, reference sequence: YP_002845730.1); 44% with β-proteobacteria Bordetella pertussis (B. per, reference sequence: NP_880910.1), and 45% with Alcaligenes faecalis (A. fae, reference sequence: WP_003803370.1); 41% with γ-proteobacteria E. coli (E. col, reference sequence: NP_417648.4), 41% with Haemophilus influenza (H. inf, reference sequence: NP_439483.1), 42% with Yersinia pestis (Y. pes, reference sequence: NP_668016.1), and 39% with Vibrio cholerae (V. cho, reference sequence: NP_230283.1). Identical residues are highlighted in red while the conserved amino acid changes are outlined in gray rectangular boxes. The conserved domain architecture of Wol GreA (typical of GreA superfamily) has two domains, N-terminal domain (6–78 a.a) enclosed in black box consisting of two α helices (red spiral), α1 (13–43 a.a) and α2 (52–75 a.a) separated by 3 10 (blue spiral) (47–49 a.a) combined together to induce nucleolytic activity of RNAP and the C terminal domain (84–163 a.a) is enclosed in green box having five β sheets (yellow arrow), β1(88–99 a.a), β2 (107–115 a.a), β3 (125–129 a.a), β4 (144–148 a.a), and β5 (153–163 a.a) verged on one side by an α-helix (131–137 a.a) shaped into a compact globular structure and executes direct binding with RNAP.

Article Snippet: The binding of Wol GreA and its domains with E. coli RNAP holoenzyme (Biotech Desk, India) was carried out as described earlier .

Techniques: Sequencing, Activity Assay, Binding Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: Wolbachia Transcription Elongation Factor “Wol GreA” Interacts with α2ββ′σ Subunits of RNA Polymerase through Its Dimeric C-Terminal Domain

doi: 10.1371/journal.pntd.0002930

Figure Lengend Snippet: (A) A computationally derived structural model of Wol GreA. The bioinformatics tool MODELLER9v10 was used to create a 3D ribbon model of Wol GreA that contained NTD (cyan) with RNA polymerase (RNAP) nucleolytic activity and CTD (red) possessing RNAP binding activity. (B) Structural superimposition of Wol GreA (red) with E. coli GreA (green) having PDB code1GRJ signifies an excellent template exhibiting 46% identity, 68% similarity, and 0.674 Å root mean square deviation (RMSD). (C) Ramachandran plot of Wol GreA constructed by PROCHECK revealed 89.8% a.a (red) residues of Wol GreA in most favored regions while 9.9% a.a (yellow) in additionally allowed regions and 0.3% a.a (pale yellow) in generously allowed regions, 0% a.a (white) residues were in disallowed regions. (D) Energy profile of Wol GreA was predicted by ProSA web server.

Article Snippet: The binding of Wol GreA and its domains with E. coli RNAP holoenzyme (Biotech Desk, India) was carried out as described earlier .

Techniques: Derivative Assay, Activity Assay, Binding Assay, Construct

Journal: PLoS Neglected Tropical Diseases

Article Title: Wolbachia Transcription Elongation Factor “Wol GreA” Interacts with α2ββ′σ Subunits of RNA Polymerase through Its Dimeric C-Terminal Domain

doi: 10.1371/journal.pntd.0002930

Figure Lengend Snippet: (A) Superdex-75 10/300 GL column pre-equilibrated with buffer (50-mM NaH 2 PO 4 , 50-mM NaCl, 0.1% NaN 3 , pH 6.5) was used for Wol GreA at a flow rate of 0.5 ml/min at 25°C. (B) Under the same conditions, a single elution peak was observed with Wol NTDat its meticulous monomeric position of ∼9-kDa. (C) Wol CTD also revealed a single elution peak of the RNAP binding domain at its dimeric position of ∼20.6-kDa under identical elution condition.

Article Snippet: The binding of Wol GreA and its domains with E. coli RNAP holoenzyme (Biotech Desk, India) was carried out as described earlier .

Techniques: Binding Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: Wolbachia Transcription Elongation Factor “Wol GreA” Interacts with α2ββ′σ Subunits of RNA Polymerase through Its Dimeric C-Terminal Domain

doi: 10.1371/journal.pntd.0002930

Figure Lengend Snippet: (A) The binding study of Wol GreA and its domains with RNAP by chemiluminescent Western blotting using column with 100-kDa cut-off membrane. Lane 1, upper fraction of reaction one collected from the column containing 2-µM Wol NTD alone in the binding buffer A (40-mM Tris-HCl pH 7.5, 30-mM KCl, 10-mM MgCl 2, 80-mM NaCl, 0.1-mM EDTA, and 0.1-mM DTT); Lane 2, upper fraction of reaction two from the column where 2-µM of Wol NTD was incubated at 4°C for 30 min with 10-U RNAP holoenzyme in the same buffer; Lane 3, upper fraction of reaction three from the column containing 2-µM Wol CTD alone in the binding buffer A; Lane 4, upper fraction of reaction four collected from the column where 2-µM of Wol CTD was incubated at 4°C for 30 min with 10-U RNAP holoenzyme in the same buffer; Lane 5, upper fraction of reaction five from column containing 2-µM Wol GreA alone in buffer A; Lane 6, upper fraction of reaction six from column where 2-µM of Wol GreA was incubated at 4°C for 30 min with 10-U RNAP holoenzyme in the same buffer. (B) Binding of Wol GreA and its domains with different subunits of RNAP was further validated on a silver stained gel. Lane M, protein marker; Lane 1, Wol NTD; Lane 2, Wol NTD and 10-U-RNAP holoenzyme; Lane 3, Wol CTD; Lane 4, Wol CTD and 10-U RNAP holoenzyme; Lane 5, Wol GreA. Lane 6, Wol GreA and 10-U RNAP holoenzyme. (C) Ponceau stained blot. Lane M, standard protein marker; Lane 1-3, 10-U of RNAP holoenzyme (α2ββ′σ subunits) were resolved followed by Ponceau staining. (D) Far Western Blotting was performed to determine the specific interaction of Wol GreA and CTD with α2ββ′σ subunits of RNAP. Lane M, protein molecular weight marker; Lane 1, blot strip with 10-U of RNAP as a prey protein; Lane 2, blot strip with 10 U of RNAP probed with 2-µM Wol GreA at 4°C in binding buffer A; Lane 3, blot strip with 10 U of RNAP probed with 2-µM Wol CTD at 4°C in binding buffer A.

Article Snippet: The binding of Wol GreA and its domains with E. coli RNAP holoenzyme (Biotech Desk, India) was carried out as described earlier .

Techniques: Binding Assay, Western Blot, Membrane, Incubation, Staining, Marker, Far Western Blot, Molecular Weight, Stripping Membranes

Journal: PLoS Neglected Tropical Diseases

Article Title: Wolbachia Transcription Elongation Factor “Wol GreA” Interacts with α2ββ′σ Subunits of RNA Polymerase through Its Dimeric C-Terminal Domain

doi: 10.1371/journal.pntd.0002930

Figure Lengend Snippet: For protein–protein docking the HEX program based on a rigid body protein docking algorithm that explicitly determines the steric shape, electrostatic potential, and charge density of the protein as expansions of spherical polar Fast Fourier Transformation (FFT) basis functions was exploited. (A) Interaction of ribbon model of α-subunit (green) of RNAP with Wol GreA consisting of NTD (cyan) and CTD (red). (B) Interaction of ribbon model β-subunit (green) of RNAP with Wol GreA having both its domains. (C) Interaction of ribbon model of β′-subunit (green) of RNAP with Wol GreA consisting of both the domains. (D) Interaction of ribbon model of σ-subunit (green) of RNAP with Wol GreA consisting of both the domains.

Article Snippet: The binding of Wol GreA and its domains with E. coli RNAP holoenzyme (Biotech Desk, India) was carried out as described earlier .

Techniques: Transformation Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: Wolbachia Transcription Elongation Factor “Wol GreA” Interacts with α2ββ′σ Subunits of RNA Polymerase through Its Dimeric C-Terminal Domain

doi: 10.1371/journal.pntd.0002930

Figure Lengend Snippet: (A) The protein docking study between α-subunit of RNAP and Wol GreA exhibited that CTD Lys140 donor atoms involved in hydrogen (H) bonding with acceptor atoms of Wol α subunit Thr164 and Wol CTD Asp120, Lys82 acceptor atoms form H bonding with Wol α subunit donor atoms, Asn50, Arg53, and Thr88. (B) The protein docking between β-subunit of RNAP and Wol GreA exhibited that CTD Ser105, Ser127, Ser129 donor atoms formed H bonding with Wol RNAP β subunit Asn528, Ser529, Asp 1330, Asp1331 acceptor atoms and Wol CTD Ser98, Glu116, Ser129, Val147 acceptor atoms created H bonding by interacting with Wol RNAP β subunit Lys163, Asn528, Ser529, Ser530, Asn589, and Arg1359 donor atoms. (C) Similar to α and β-subunits of RNAP, β′ also solely forms H bonding with CTD residues of Wol GreA where Ser151 donor atom formed H bond with Leu1178 acceptor atom of Wol RNAP β′ subunit and its acceptor atoms Asp104, Tyr109, Val121, Ser122, Glu153, and Phe163 involved in binding with donor atoms of Wol RNAP β′ subunit Arg129, Arg1135, Lys1195, Arg1211, Gly1297, and Arg1300. (D) The protein docking between σ-subunit of RNAP and Wol GreA exhibited that CTD acceptor atoms Glu106, Glu143 created H bonding by interacting with Lys430, Arg478 donor atoms of RNAP σ subunit.

Article Snippet: The binding of Wol GreA and its domains with E. coli RNAP holoenzyme (Biotech Desk, India) was carried out as described earlier .

Techniques: Binding Assay