Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/chemistry , DNA/metabolism , Saccharomyces cerevisiae/genetics , Binding Sites , Cloning, Molecular/methods , DNA-Binding Proteins/genetics , Genes, Reporter , Mutagenesis, Site-Directed , Plasmids , Polymerase Chain Reaction/methods , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombination, Genetic , Sequence Analysis, Protein/methods , beta-Galactosidase/analysis , beta-Galactosidase/geneticsABSTRACT
We describe a method for the genetic analysis of the DNA-binding properties of Xenopus transcription factor IIIA (TFIIIA). In this approach, a transcriptional activator with the DNA-binding specificity of Xenopus TFIIIA is expressed in yeast cells, where it specifically activates expression of a beta-galactosidase reporter gene containing one or more Xenopus 5 S rRNA genes that function as upstream activator sequences. This transcription-promoting activity was used as the basis for a genetic assay of Xenopus TFIIIA's DNA-binding function in yeast, an assay that we show can be calibrated quantitatively to allow the affinity of the Xenopus TFIIIA-5 S rRNA gene interaction to be deduced from measurements of beta-galactosidase activity. We have combined this genetic assay with a simple and efficient method of mutagenesis that makes use of error-prone PCR and homologous recombination to generate and screen large numbers of TFIIIA mutants for those with altered 5 S rRNA gene-binding affinity. Over 30 such mutants have been identified and partially characterized. The mutants we have obtained provide strong support for the application to intact TFIIIA of recent structural models of the N-terminal zinc fingers of the protein bound to fragments of the 5 S rRNA gene. Other mutants permit identification of important residues in more C-terminal zinc fingers of TFIIIA for which high-resolution structural information is not currently available. Finally, our results have interesting implications with respect to the mechanism of activation of transcription by RNA polymerase II in yeast.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA/metabolism , Genetic Testing , RNA, Ribosomal, 5S/genetics , RNA, Ribosomal, 5S/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Base Sequence , Molecular Sequence Data , Mutagenesis , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Transcription Factor TFIIIA , Xenopus , Zinc Fingers/genetics , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
An acid phosphatase activity (APA; EC 3.1.3.2) was demonstrated in homogenates of adult Hymenolepis diminuta. The APA was soluble based on the observation that it did not sediment at 130,000 g. APA was partially purified using a combination of differential centrifugation, ammonium sulphate precipitation, chloroform extraction, and gel and fast-protein-liquid-chromatography. This combination of techniques resulted in a preparation with a specific activity approximately 500 times greater than the crude enzyme preparation. The temperature and pH optima of the partially purified APA were 44 degrees C and pH 5.0. The enzyme appeared to be a monomer with a molecular weight of approximately 62,000. APA had a higher affinity for a greater activity towards aromatic than aliphatic phosphoesters, and phosphoryl transferase activity was demonstrable using 1-butanol and ethylene glycol as acceptors. APA was inhibited significantly by sodium dodecyl sulphate, fluoride, molybdate and tartrate, but CuSO4 and Fast Garnet GBC were poor inhibitors. The precise cellular localization and function of this enzyme remains unknown since it possesses characteristics of both cytoplasmic and lysosomal APA's of other organisms.
