The developmental control of the expression of Xenopus 5S rRNA genes is mediated by the activitiy of an exceptional protein called transcription factor IIIA (TFIIIA). This protein has the distinctive ability to bind to both the 5S gene and its transcript, 5S rRNA. TFIIIA as well as the whole collection of proteins that control the activity of oocyte and somatic 5S genes are the prototype for the study of developmental regulation of gene expression and determination. The identification of the binding site for TFIIIA on 5S rRNA has led us to propose that the protein utilizes similar contacts in binding to either nucleic acid. We will test this proposal in two ways. Using circular dichroism spectroscopy we will determine the conformation of the DNA when TFIIIA binds to the 5S gene. If the interaction of the protein with the two nucleic acids is similar then the DNA will have to be in the A conformation in order to form a structure comparable to 5S rRna. We will carry out an extensive series of site-directed mutagenesis experiments to make changes in both the gene and the transcript. We will determine whether a particular change has a similar effect on the binding of TFIIIA to both nucleic acids. The binding site on 5S rRNA for TFIIIA closely approximates that for ribosomal protein L5. This suggests that the two proteins may be related; at least there may be homology between their nucleic acid binding domains. We will measure the binding of L5 and TFIIIA to the variant 5S rRNAs synthesized in the mutagenesis experiments to compare the interactions of the proteins with their cognate nucleic acid. We will test the homology of the proteins by immunochemical techniques. We will prepare polyclonal antibodies to a proteolytic fragment of TFIIIA that contains the nucleic acid binding domain of the protein. We will determine whether the antiserum crossreacts with L5. If the result is positive, we will use the antiserum to locate the nucleic acid binding domain within L5. The unique properties of TFIIIA allow us to make certain invaluable comparisons: between the structures of two nucleic acids that are recognized by this protein, and between two proteins that recognize the same binding site on 5S rRNA.
