Nucleic Acids Research, 1989, Vol. 17, No. 23 9861-9870
© 1989
MOLECULAR BIOLOGY |
Internal deletion mutants of Xenopus transcription factor IIIA
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center Oklahoma City, OK 73190, USA
*To whom correspondence should be addressed
Received May 17, 1989. Revised October 20, 1989. Accepted October 20, 1989.
Xenopus transcription factor IIIA (TFIIIA) or TFIIIA mutants with internal deletions were expressed in E. coli utilizing a bacteriophage T7 RNA polymerase system. TFIIIA or deletion mutant TFIIIAs, isolated from E.coli cell extracts, were identified by SDS PAGE and immunoblotting with rabbit antiserum against native TFIIIA purified from Xenopus immature oocytes. Specific DNA binding of intact or internally deleted TFIIIA was compared by analyzing their abilities to protect the internal control gene (ICR) of the Xenopus 5S RNA gene from DNase I digestion. Intact protein, synthesized from a full-length TFIIIA eDNA, bound specifically to the entire ICR (+96 to +43) and promoted 5S RNA gene transcription in vitro. One TFIIIA deletion mutant, expressed from eDNA lacking the coding sequence for the putative fourth zinc finger (designated from the N-terminus, amino acids 103–132) protected the ICR from DNase I digestion from nucleotide positions +96 to +78. A second TFIIIA mutant resulting from fusion of putative zinc fingers 7 and 8 (deletion of amino acids 200+224) protected the 5S gene ICR from positions +96 to +63. The DNase I protection patterns of these mutant proteins are consistent with the formation of strong ICR contacts by those regions of the protein on the N- terminal side of the mutation but not by those regions on the C-terminal side of the mutation. The regions of the protein comprising the N-terminal 3 fingers and N-terminal six fingers appear to be in contact with approximately 18 and 33 bp of DNA respectively on the 3' side of the 5S gene ICR . These internal deletion mutants promoted 5S RNA synthesis in vitro and DNA renaturation.
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