TFIIIA induced DNA bending: effect of low ionic strength electrophoresis buffer conditions.
We have used a circular permutation gel shift assay to show that the 5S gene transcription factor, TFIIIA, induces a bend at the internal promoter of the Xenopus oocyte-type 5S gene. The degree of bending is comparable to what we have previously observed for TFIIIA induced bending of the Xenopus somatic-type gene [Schroth, G.P. et al. (1989) Nature 340, 487-488]. In addition, we show that TFIIIA induced DNA bending is dramatically affected by the ionic conditions used during gel electrophoresis. By modifying the conditions of the electrophoresis, we can detect two distinct conformations for the TFIIIA/DNA complex. In very low ionic strength buffers, the degree of DNA bending in the complex is estimated to be about 25 to 30 degrees, whereas in higher ionic strength buffers it is about 60 to 65 degrees. These data explain the apparent discrepancy between our results and the results of another study in which it was claimed that TFIIIA did not 'substantially' bend DNA [Zweib, C. and Brown, R.S. (1990) Nucleic Acid Res. 18, 583-587]. These results also demonstrate that the TFIIIA/DNA complex has a large degree of conformational flexibility. Both DNA bending and conformational flexibility are structural features which may provide a key insight into the function of TFIIIA as a positive transcription factor.
Schroth
G P
GP
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Gottesfeld
J M
JM
Bradbury
E M
EM
D000818:Animals; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D066298:In Vitro Techniques; D009690:Nucleic Acid Conformation; D009865:Oocytes; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012341:RNA, Ribosomal, 5S / Q000235:genetics*; D015183:Restriction Mapping; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000378:metabolism*; D014982:Xenopus laevis
D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors