Transition Mutations within the Xenopus borealis Somatic 5S RNA Gene Can have Independent Effects on Transcription and TFIIIA Binding

Abstract

Base-pair changes were introduced into the Xenopus borealis somatic 5S RNA gene by treatment with sodium bisulfite. Mutants were screened by sequence determination. The collection of mutants permitted a detailed investigation of the fine structure of the intragenic control region that binds the transcription factor TFIIIA. Selected mutants were recloned in tandem with a somatic 5S RNA maxigene to permit sensitive measurement of their relative transcription activities. The transcription efficiencies of a number of mutations at the borders of the control region were correlated with TFIIIA binding by using DNase I protection (footprinting) assays. Mutations affecting transcription and TFIIIA binding extended from gene residues 46 to 91. The results reinforce a model in which the distal half of the protected region constitutes a tight binding domain for TFIIIA. A number of transitions in the 5′ domain led to significant increases or decreases in transcription efficiency, but resulted in barely detectable changes in TFIIIA binding. Two mutants with C→T transitions at gene residues 52 and 53 were transcribed with increased efficiencies (up phenotype). These results suggest that TFIIIA must make appropriate contacts within the 5′ domain of the control region to permit subsequent binding of other factors in stable transcription complexes.