Domains of the positive transcription factor specific for the Xenopus 5S RNA gene

Abstract

The 40 kd positive transcription factor that interacts with the 50 nucleotide internal control region of 5S ribosomal RNA genes in Xenopus can be subdivided into three functional domains by proteolytic cleavage. At one end of the protein is a 10 kd domain that is required for efficient RNA transcription but not for binding to the DNA. This adjoins a second domain that binds to the 5' end of the internal control region, a region of the DNA known to contribute only weakly to binding the protein but one that is essential for RNA transcription. The removal of both of these protein domains leaves a half of the protein that binds only to the 3' side of the control region and is inactive in promoting transcription. Quantitative DNA binding and in vitro transcription experiments show that only a single molecule of 40 kd factor binds to the internal control region, and that this stoichiometry is sufficient to give maximal stimulation of 5S RNA transcription in a reconstituted system. Consideration of the essential DNA contact points of the factor, taken together with the constraints imposed by a 1:1 protein to DNA stoichiometry permit the linear projection of the extended protein molecule onto its binding site along the internal control region of the 5S RNA gene.