High yield purification of active transcription factor IIIA expressed in E. coli.
Transcription factor IIIA (TFIIIA), a sequence-specific DNA-binding protein from Xenopus laevis, is a zinc finger protein required for transcription of 5S rRNA genes by RNA polymerase III. We describe the purification and characterization of recombinant TFIIIA (recTFIIIA) expressed in E. coli. RecTFIIIA was purified to greater than 95% homogeneity at a yield of 2-3 milligrams per liter of bacterial culture. This purified protein protects the internal control region of a 5S rRNA gene from DNase I digestion, yielding footprints on both strands identical to those produced by the ovarian protein (ovaTFIIIA). Quantitative analysis of binding data from gel retardation assays yielded a KD of about 0.4 nM for TFIIIA from either source. Using a quantitative TFIIIA-dependent in vitro transcription assay, we found that recTFIIIA is equivalent to ovaTFIIIA in supporting transcription of 5S rRNA genes. We conclude that recTFIIIA is functionally indistinguishable from the protein purified from Xenopus ovaries, and can be readily obtained in pure form and large quantity.
Del Río
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Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, OH 44106.
Setzer
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000302:isolation & purification*; D014158:Transcription, Genetic; D014982:Xenopus laevis
D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I