Journal: PLoS Pathogens

Article Title: The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

doi: 10.1371/journal.ppat.1008843

Figure Lengend Snippet: Full-length WT or 3L_A mutants, which mutates the conserved RLLLG motif, of Strep-tagged ORF24 (A) or homologs from MHV68 (mu24) (B), EBV (BcRF1) (C), and HCMV (UL87) (D) were transiently transfected into HEK293T cells then co-affinity purified (AP) with StrepTactinXT beads followed by western blotting. (*) indicates the presence of a non-specific band seen while using the anti-Strep antibody.

Article Snippet: To generate the plasmid for MBP-ORF24-NTD WT (Addgene #138465) and 3L_A (Addgene #138466) expression, ORF24-NTD was PCR amplified from pcDNA4/TO-ORF24-3xFLAG WT (Addgene #138423) or 3L_A (Addgene #138425) [ ] with primers to introduce SacI and BamHI sites and cloned into pMAL-c2X using T4 DNA ligase. pMAL-c2X encodes an N-terminal MBP tag and the plasmids were cloned to express ORF24-NTD with a noncleavable MBP tag.

Techniques: Transfection, Affinity Purification, Western Blot

Journal: PLoS Pathogens

Article Title: The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

doi: 10.1371/journal.ppat.1008843

Figure Lengend Snippet: (A) Schematic of constructs used to identify a minimal N-terminal domain of ORF24 showing the predicted boundaries for the N-terminal domain, the TBP-like domain, and the C-terminal domain, including residues known to be required for Pol II binding (amino acids 73–75) and interaction with ORF34 (amino acid 328). (B) HEK293T cells were transiently transfected with full-length or truncated FLAG-tagged ORF24 and co-immunoprecipitated (IP) with anti-FLAG beads followed by western blotting with the indicated antibodies to detect ORF24 and Pol II. (*) indicates the presence of a non-specific band seen while using the anti-Strep antibody.

Article Snippet: To generate the plasmid for MBP-ORF24-NTD WT (Addgene #138465) and 3L_A (Addgene #138466) expression, ORF24-NTD was PCR amplified from pcDNA4/TO-ORF24-3xFLAG WT (Addgene #138423) or 3L_A (Addgene #138425) [ ] with primers to introduce SacI and BamHI sites and cloned into pMAL-c2X using T4 DNA ligase. pMAL-c2X encodes an N-terminal MBP tag and the plasmids were cloned to express ORF24-NTD with a noncleavable MBP tag.

Techniques: Construct, Binding Assay, Transfection, Immunoprecipitation, Western Blot

Journal: PLoS Pathogens

Article Title: The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

doi: 10.1371/journal.ppat.1008843

Figure Lengend Snippet: (A-D) Full-length or truncated Strep-tagged constructs of ORF24. (A) or homologs from MHV68 (mu24; B), EBV (BcRF1; C), and HCMV (UL87; D) were transiently transfected into HEK293T cells then co-affinity purified (AP) with StrepTactinXT beads followed by western blotting. (*) indicates the presence of a non-specific band seen while using the anti-Strep antibody.

Article Snippet: To generate the plasmid for MBP-ORF24-NTD WT (Addgene #138465) and 3L_A (Addgene #138466) expression, ORF24-NTD was PCR amplified from pcDNA4/TO-ORF24-3xFLAG WT (Addgene #138423) or 3L_A (Addgene #138425) [ ] with primers to introduce SacI and BamHI sites and cloned into pMAL-c2X using T4 DNA ligase. pMAL-c2X encodes an N-terminal MBP tag and the plasmids were cloned to express ORF24-NTD with a noncleavable MBP tag.

Techniques: Construct, Transfection, Affinity Purification, Western Blot

Journal: PLoS Pathogens

Article Title: The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

doi: 10.1371/journal.ppat.1008843

Figure Lengend Snippet: (A) Colloidal Coomassie gel demonstrating that GST and GST-ORF24-NTD can be recombinantly expressed in E. coli and purified by glutathione sepharose purification. (B) GST or GST-ORF24-NTD was incubated in HEK293T whole cell lysate, then subjected to affinity purification using glutathione magnetic beads (GSH AP) followed by western blotting. (C) Sequential reconstitution strategy for a minimal PIC containing GST-ORF24-NTD. (D) Representative reference-free two-dimensional class averages of negatively stained minimal PICs (TBP/TFIIA/TFIIB/TFIIF/Pol II/DNA) assembled in the presence of GST-ORF24-NTD. Three classes on the left show different views of the minimal PIC alone, with the class average in the upper-left annotated with the main features of a minimal PIC particle. The nine class averages on the right show diffuse density in various positions around the Pol II stalk attributed to bound GST-ORF24-NTD (green arrows). (E) Representative three-dimensional class averages of negatively stained minimal PICs assembled in the presence of GST-ORF24-NTD. Classes 1 and 2 exhibit two major areas occupied by bound GST-ORF24-NTD proximal to the Pol II stalk, while class 5 does not exhibit any such density near the Pol II stalk. Solid surfaces are colored by subunit, while a lower intensity iso-surface is shown in transparency to reveal the weaker density attributed to bound GST-ORF24-NTD (green arrows). (F) Difference mapping of the densities attributed to bound GST-ORF24-NTD. Shown on the left are two-dimensional projections of class 1 (top) and 2 (bottom) from (E), and on the right are the difference maps, called “Class 1 Δ” and “Class 2 Δ”, calculated by subtracting Class 5 from each of the respective classes. (G) Three-dimensional difference maps corresponding to the extra density attributed to bound GST-ORF24-NTD, mapped onto the structure of the minimal PIC (PDB 5IYA). (H) Zoomed in view of (G) with the structure of Schizosaccharomyces pombe Rpb1 (PDB 3H0G) superposed onto the human structure to show the location of the beginning portion of the Rpb1 CTD within the Pol II stalk. Note that only the very N-terminal portion of the Rpb1 CTD is visible in this structure, with >450 amino acids following this sequence in the CTD of human Rpb1.

Article Snippet: To generate the plasmid for MBP-ORF24-NTD WT (Addgene #138465) and 3L_A (Addgene #138466) expression, ORF24-NTD was PCR amplified from pcDNA4/TO-ORF24-3xFLAG WT (Addgene #138423) or 3L_A (Addgene #138425) [ ] with primers to introduce SacI and BamHI sites and cloned into pMAL-c2X using T4 DNA ligase. pMAL-c2X encodes an N-terminal MBP tag and the plasmids were cloned to express ORF24-NTD with a noncleavable MBP tag.

Techniques: Purification, Incubation, Affinity Purification, Magnetic Beads, Western Blot, Staining, Sequencing

Journal: PLoS Pathogens

Article Title: The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

doi: 10.1371/journal.ppat.1008843

Figure Lengend Snippet: (A) Recombinantly purified GST-CTD repeats or the GST-CTD linker were incubated with either purified MBP-ORF24-NTD or MBP-ORF24-NTD-3L_A protein, then subjected to a glutathione pulldown (GSH AP). Samples were resolved by SDS-PAGE and analyzed by western blot. (B) Recombinantly purified GST-CTD repeats or GST-Rpb4/His-Rpb7 heterodimer were incubated with recombinantly purified Strep-tagged ORF24-NTD, then subjected to a StrepTactinXT pulldown (AP). Samples were resolved by SDS-PAGE and analyzed by western blot. (C) Recombinantly purified full-length GST-CTD repeats (52 repeats) shorter GST-CTD constructs (10, 4, or 2 repeats), or GST itself were incubated with recombinantly purified Strep-tagged ORF24-NTD, then subjected to a StrepTactinXT pulldown (AP). Samples were resolved by SDS-PAGE and stained with colloidal Coomassie.

Article Snippet: To generate the plasmid for MBP-ORF24-NTD WT (Addgene #138465) and 3L_A (Addgene #138466) expression, ORF24-NTD was PCR amplified from pcDNA4/TO-ORF24-3xFLAG WT (Addgene #138423) or 3L_A (Addgene #138425) [ ] with primers to introduce SacI and BamHI sites and cloned into pMAL-c2X using T4 DNA ligase. pMAL-c2X encodes an N-terminal MBP tag and the plasmids were cloned to express ORF24-NTD with a noncleavable MBP tag.

Techniques: Purification, Incubation, SDS Page, Western Blot, Construct, Staining