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The yeast transcription factor TFIIIB has been examined with regard to its hydrodynamic properties. This protein, which is required for both tRNA and 5 S rRNA gene transcription in vitro, exhibits unusual physical properties. When analyzed by denaturing polyacrylamide gel electrophoresis TFIIIB is a single polypeptide of Mr approximately 60,000. When this transcription factor is subjected to rate zonal centrifugation on glycerol gradients, it sediments between ovalbumin (Mr = 45,000) and bovine serum albumin (Mr = 66,300). This sedimentation behavior is consistent with TFIIIB being a monomer in its native state. However, when TFIIIB was analyzed by gel filtration chromatography it was determined to have a Stokes radius of 53 A, eluting from this chromatography matrix near the position of catalase (Mr = 248,000). This anomalous behavior suggests that TFIIIB is a very asymmetric molecule. Purified TFIIIB was subjected to amino acid analyses and the resulting composition data used to calculate a partial specific volume for the intact molecule. All of these data were then used to estimate possible molecular dimensions of the native TFIIIB molecule. Results of these calculations are consistent with the idea that this transcription factor is a very elongated entity.