Rapid Enrichment of HeLa Transcription Factors IIIB and IIIC by Using Affinity Chromatography Based on Avidin-Biotin Interactions

Abstract

Plasmid DNA containing the adenovirus type 2 genes for VA RNA was linearized at a site distal to the gene, end labeled with a biotin-nucleotide analog of TTP, and incubated with avidin to form an avidin-biotinylated DNA complex. HeLa cell S100 extracts containing crude RNA polymerase III and transcription factors (TFs) IIIB and IIIC were programmed with the avidin-biotin-VA DNA to allow stable complex formation (A.B. Lassar, P.L. Martin, and R.G. Roeder, Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-cellulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCl to remove RNA polymerase III and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCl. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCl; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCl elution. The specific activity of the TFIIIC in the 3.0 M KCl fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCl fractions was reduced 7,500- and 100,000-fold, respectively.

Purification of Sequence‐Specific DNA‐Binding Proteins by Affinity Chromatography

“…Other methods have been described to purify sequence‐specific DNA‐binding proteins, including chromatography using biotinylated DNA fragments attached to various supports by biotin‐avidin or biotin‐streptavidin coupling ( UNIT here; Chodosh et al, ; Kasher et al, ; Leblond‐Francillard et al, ), oligonucleotides synthesized onto Teflon‐based beads (Duncan and Cavalier, ), or synthetic oligonucleotide monomers attached to agarose supports (Wu et al, ; Blanks and McLaughlin, ; Hoey et al, ); and preparative gel mobility shifts (Gander et al, ). Although more than 50 sequence‐specific DNA‐binding proteins have been purified by the method described in this unit (Kadonaga, , and references therein), it is likely that many of the techniques listed above are also effective for purifying sequence‐specific DNA‐binding proteins.…”