rsp5 expression  (Addgene inc)

Journal:

Article Title: The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase

doi:

Figure Lengend Snippet: (A) Generation of the GAL-RSP5 strain. Nucleotides 1–1,077 of the RSP5 gene (rsp5-Δ) were cloned into the pYES2 vector (Invitrogen) lacking a 2-μm replication origin. This was used to transform the FY56 yeast strain (ura3-52), selecting for growth on 2% galactose plates in the absence of uracil. The predicted recombination product, which was confirmed by PCR analysis of genomic DNA, is shown. (B) The RSP5 and GAL-RSP5 strains were grown at 30°C in minimal medium with 2% galactose to an OD600 of 0.5. Aliquots of the culture were removed to either fresh galactose- or dextrose-containing medium and incubated for an additional 36 h. Total cell extracts were analyzed by SDS/PAGE and immunoblotting with antibodies against Rpb1, Rsp5, and the 70-kDa subunit of yeast replication protein A (RPA). Extracts from the RSP5 strain, grown in galactose (Gal.) and dextrose (Dex.), respectively, were analyzed in lanes 1 and 2, and extracts from the GAL-RSP5 strain were analyzed in lanes 3 and 4. Rsp5 protein from the GAL-RSP5 strain migrates slightly slower due to a 12-amino acid epitope at its amino terminus.

Article Snippet: Recombinant baculoviruses expressing Rsp5 or Rsp5 C-A proteins were generated using the BaculoGold system (PharMingen).

Techniques: Clone Assay, Plasmid Preparation, Incubation, SDS Page, Western Blot

Journal:

Article Title: The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase

doi:

Figure Lengend Snippet: Binding and ubiquitination of 35S-labeled yeast proteins by Rsp5. Glutathione-Sepharose-bound GST-Rsp5 fusion proteins, either wild type (lanes 1–3) or the C-A mutant (lanes 4–6), were incubated with 35S-labeled total yeast extract. The Sepharose beads were collected, washed, and then incubated without (lanes 1 and 4) or with (lanes 2, 3, 5, 6) recombinant E1 enzyme and UBC8 (lanes 2 and 5) or UBC1 (lanes 3 and 6) E2 protein. Proteins were then denatured in loading buffer and analyzed by SDS/PAGE and autoradiography. Three protein species that appear to be bound and ubiquitinated specifically by wild-type GST-Rsp5/UBC8 are indicated at left (p200, p160, p60), and migration positions of molecular weight markers are indicated at right.

Article Snippet: Recombinant baculoviruses expressing Rsp5 or Rsp5 C-A proteins were generated using the BaculoGold system (PharMingen).

Techniques: Binding Assay, Labeling, Mutagenesis, Incubation, Recombinant, SDS Page, Autoradiography, Migration, Molecular Weight

Journal:

Article Title: The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase

doi:

Figure Lengend Snippet: The FY56 strain was transformed with the pYES2 vector or the vector expressing GST, GST-Rsp5, or GST-Rsp5 N terminus (lanes 1–4, respectively). Extracts were made from the cells and glutathione binding proteins, as well as copurifying proteins, were isolated on glutathione-Sepharose and analyzed by SDS/PAGE and immunoblotting. GST (nonfusion protein) expression was detected separately by silver staining of the glutathione-bound fraction (data not shown).

Article Snippet: Recombinant baculoviruses expressing Rsp5 or Rsp5 C-A proteins were generated using the BaculoGold system (PharMingen).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Binding Assay, Isolation, SDS Page, Western Blot, Silver Staining

Journal:

Article Title: The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase

doi:

Figure Lengend Snippet: (A) Binding of 35S-labeled in vitro-translated Rpb1 to GST (−, no fusion), or GST fused to E6-AP, Rsp5, the Rsp5 C-A mutant, the hect domain of Rsp5, or the N-terminal domain of Rsp5 (lanes 1–6, respectively). Five microliters of the translation reaction was used in the binding assays and was also loaded in lane 7. (B) Binding of carboxyl-terminally truncated Rpb1 to Rsp5. The Rpb1 gene in pYES2 was digested with BsiWI (nucleotide 4,528 of the 5,181 nucleotide ORF). The full-length and truncated genes were used in in vitro transcription/translation reactions. One microliter of the translation reactions was run in lanes 3 and 4. (C) Binding of Rsp5 to GST-CTD. In vitro-translated Rsp5, the Rsp5 hect domain, the Rsp5 amino-terminal domain (N-term), and E6-AP were assayed for binding to GST-CTD (lanes 1–4, respectively). One microliter of the translation products was run in lanes 5–8.

Article Snippet: Recombinant baculoviruses expressing Rsp5 or Rsp5 C-A proteins were generated using the BaculoGold system (PharMingen).

Techniques: Binding Assay, Labeling, In Vitro, Mutagenesis

Journal:

Article Title: The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase

doi:

Figure Lengend Snippet: (A) In vitro ubiquitination of Rpb1. In vitro-translated Rpb1 was incubated without (−, lane 1) or with a DEAE high-salt fraction derived from insect cells infected with nonrecombinant baculovirus (wtv, lane 2), Rsp5 virus (lane 3), or the C-A mutant (lane 4), along with recombinant E1 and E2 (UBC8) proteins, ATP, and ubiquitin. The migration position of full-length Rpb1 is indicated, as is multi-ubiquitinated Rpb1 [Rpb1-ub(n)]. (B) Ubiquitination of GST-CTD. In vitro-translated GST (lanes 1–3) or GST-CTD (lanes 4–6) were incubated with a control protein fraction (lanes 1 and 4), Rsp5 (lanes 2 and 5), or Rsp5 C-A (lanes 3 and 6), as in A. The ubiquitinated GST-CTD species are indicated.

Article Snippet: Recombinant baculoviruses expressing Rsp5 or Rsp5 C-A proteins were generated using the BaculoGold system (PharMingen).

Techniques: In Vitro, Incubation, Derivative Assay, Infection, Mutagenesis, Recombinant, Migration

Journal:

Article Title: The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase

doi:

Figure Lengend Snippet: (A) Purified pol II from HeLa cells was assayed for binding to GST-E6-AP and Rsp5 (lanes 2 and 3). The largest and second largest subunits were detected by immunoblotting. The input amount of protein was loaded in lane 1. The phosphorylated and underphosphorylated forms of the largest subunit are indicated. (B) In vitro ubiquitination of human pol II large subunit. Total HeLa cell extract was incubated without (−, lane 1) or with a DEAE fraction from insect cells infected with nonrecombinant baculovirus (wtv, lane 2), Rsp5-expressing virus (Rsp5, lane 3), or Rsp5 C-A-expressing virus, along with ubiquitin, ATP, and E1 and UBC8 E2 proteins. The reactions were analyzed SDS/PAGE and immunoblotting with the anti-CTD antibody.

Article Snippet: Recombinant baculoviruses expressing Rsp5 or Rsp5 C-A proteins were generated using the BaculoGold system (PharMingen).

Techniques: Purification, Binding Assay, Western Blot, In Vitro, Incubation, Infection, Expressing, SDS Page