Nucleic Acids Research, 1994, Vol. 22, No. 3 443-449
© 1994
MOLECULAR BIOLOGY |
Bead-shift isolation of protein-DNA complexes on a 5S RNA gene
Sinsheimer Laboratories, University of California Santa Cruz, CA 95064, USA
Received September 10, 1993. Accepted December 19, 1993.
Specific protein-DNA complexes formed on a Xenopus 5S RNA gene were Isolated and characterized using a novel technique. A DNA template reversibly immobilized on paramagnetic beads was used to capture, affinity purify, and concentrate protein - DNA complexes formed in a whole cell extract. The complexes were then released from the beads in a soluble and transcriptionally active form via restriction enzyme digestion of the DNA. A band-shift gel was used to separate and obtain the DNase I footprints of five individual complexes. Three of the complexes resulted from the independent binding of two proteins, TFIIIA and an unidentified protein binding to a large region just downstream of the 3' end of the gene. Two more slowly migrating complexes contained an additional large central protected region covering most of the gene. The most slowly migrating complex displayed protein interactions over the 5' flanking sequences. The formation of two of these complexes was shown to be dependent on TFIIIC activity. The correlation between transcriptlonal activity and the formation of these complexes suggests that the observed protein - DNA Interactions are important for transcription of 5S RNA genes.