METHODS FOR STUDYING PROTEIN-NUCLEIC ACID INTERACTION

Abstract

ABSTRACT Attention is focused on the study of regulatory proteins, repressors or activators, which bind DNA to inhibit or stimulate transcription, respectively. The best characterized examples of repressors and activators, isolated from bacteria or phages, are cited. Some general properties are pointed out, which appear to be common to all proteins binding DNA with great specificity and high affinity. The techniques available for their purification and fractionation are mentioned. Sensitive methods for the study of protein-nucleic acid interaction involve the separation of the radioactively labelled nucleoprotein complex from the free labelled reactant. Different techniques that can be used are briefly outlined. The membrane filter technique, which is most sensitive, convenient and fast, is discussed in detail. The membrane filter technique is illustrated by its use in the study of the E. coli lactose operon repressor-operator interaction. The binding constant for that interaction is Kd = 10−13m. The effects of a variety of parameters on the binding, as well as the mode of action of inducers and anti-inducers, are known. The rate constants for the repressor-operator association and dissociation have been measured. Repressors altered by mutations have been characterized. The membrane filter technique also provides an assay and a means to purify operator DNA. The approach to similar studies in an animal cell system is briefly discussed.