Journal: The Journal of biological chemistry

Article Title: Protease footprinting analysis of ternary complex formation by human TFIIA.

doi: 10.1074/jbc.272.2.1180

Figure Lengend Snippet: FIG. 1. Experimental design. A, specific 32P-end labeling of TFIIA. Recombinant TFIIA subunits bearing a heart muscle kinase phospho- rylation site (HMK tag) at one terminus and a His-tag at the other were overexpressed and purified from Escherichia coli by Ni-NTA chroma- tography. The tagged subunits were then joined with a partner lacking the HMK tag and labeled with [g-32P]ATP and heart muscle kinase to generate TFIIA 32P-end-labeled at the HMK tag of one subunit. B, schematic of protease footprinting protocol. To form ternary complexes, recombinant human TBP (the carboxyl-terminal 181 amino acids) and human 32P-end-labeled TFIIA were incubated with a biotinylated oli- gonucleotide containing the TATA box from the adenovirus E4 pro- moter immobilized on streptavidin:magnetic beads. (Binary complexes were assembled by incubating glutathione-agarose-immobilized GST- TBP with human 32P-end-labeled TFIIA, not shown.) The complexes were washed with an excess of binding buffer to remove unbound protein and then digested with limiting amounts of a broad specificity protease. The products were fractionated on high resolution SDS-poly- acrylamide gels and visualized by autoradiography. C, specific ternary complex formation. Increasing amounts of TFIIA end-labeled on the g subunit were incubated with biotinylated TATA box oligonucleotides immobilized on M-280 streptavidin resin with (lanes 3, 5, 7, and 9) or without (lanes 2, 4, 6, and 8) TBP and then washed extensively. The bound fraction was electrophoresed on standard SDS-polyacrylamide gels and autoradiographed. Lane 1 is 3% of the input end-labeled TFIIA for the experiments shown in lanes 8 and 9.

Article Snippet: Complex Formation and Proteolysis—TBP-TFIIA-TATA box ternary complexes were formed in a 50-ml mixture containing 3 pmol of biotinylated adenovirus E4 DNA template, a synthetic biotinylated oligonucleotide (biotin-59-GGATCCCCAGTCCTATATATACTCGCTCTGC-39) immobilized on streptavidin-conjugated M-280 magnetic beads (Dynal), with 500 ng of TBP, and 400 ng of 32P-labeled TFIIA.

Techniques: End Labeling, Recombinant, Purification, Labeling, Footprinting, Incubation, Magnetic Beads, Binding Assay, Autoradiography

Journal: Cell reports

Article Title: Functional elements of the cis -regulatory lincRNA-p21

doi: 10.1016/j.celrep.2022.110687

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 50 μg of total RNA was biotinylated using MTSEA biotin-XX (Biotium) and enriched using streptavidin as previously described ( Schofield et al., 2018 ).

Techniques: Recombinant, Western Blot, Hybridization, Plasmid Preparation, Protease Inhibitor, Magnetic Beads, PCR Cloning, Software