Novel method for identifying sequence-specific DNA-binding proteins.
We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D004275:DNA, Ribosomal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D001616:beta-Galactosidase / Q000378:metabolism
D004274:DNA, Recombinant; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004262:DNA Restriction Enzymes; D001616:beta-Galactosidase