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Nucleic Acids Research, 1990, Vol. 18, No. 3 583-587
© 1990

MOLECULAR BIOLOGY

Absence of substantial bending in Xenopus laevis transcription factor IIIA-DNA complexes

Christian Zwieb* and Raymond S. Brown1

Department of Molecular Biology, The University of Texas Health Center PO Box 2003, Tyler, TX 75710, USA 1Department of Microbiology and Molecular Genetics, Harvard Medical School and Howard Hughes Medical Institute, Harvard University 7 Divinity Avenue, Cambridge, MA 02138, USA

* To whom correspondence should be addressed

Received September 29, 1989. Revised January 2, 1990. Accepted January 2, 1990.

The extent and location of DNA-bending induced in the Xenopus laevis transcription factor IIIA-oocyte 5S RNA gene complex was determined by the gel retardation method. The electrophoretic mobilities of TFIIIA complexed with restriction fragments of 160, 177, 282 and 300 bp that contain the sequence of the major oocyte 5S RNA gene were compared. In these fragments the 120-bp gene is positioned either in the middle or at the end. Minor differences in the mobility of the complexes indicate that the degree of DNA bending is only slight. To determine the bending angle more precisely, a bending vector system, pBend3, was used to examine the complex of TFIIIA with the internal control region (ICR) of the 5S RNA gene. A 61-bp synthetic duplex corresponding to the ICR sequence was cloned into pBend3. Duplicated circular permuted restriction sites allow several 186-bp fragments to be generated in which the position of the ICR can be varied. Gel retardation of TFIIIA-DNA complexes with the ICR sequence contained in pBend3 indicates a bending angle of only 30 degrees and shows that interaction in the ICR could account for all of the bending found in the complete oocyte 5S RNA gene.


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