RESUMO
Xenopus transcription factor IIIA (TFIIIA) is expressed in Escherichia coli by utilizing one plasmid with a T7 RNA polymerase gene and another plasmid with TFIIIA cDNA cloned downstream of a T7 promoter. Wild-type TFIIIA and a TFIIIA deletion mutant, isolated from E. coli cell extracts, are identified by antiserum against native TFIIIA purified from Xenopus immature oocytes. DNase I protection experiments indicate that wild-type TFIIIA, synthesized from a full-length TFIIIA cDNA, binds specifically to the coding and noncoding strands of the 5 S RNA gene. The TFIIIA deletion mutant, expressed from TFIIIA cDNA lacking the coding sequence for the N-terminal 29 amino acids, fails to bind specifically to the 5 S RNA gene as judged by its inability to protect to any degree the coding or noncoding strands of the gene from DNase I digestion. Both wild-type TFIIIA and the N-terminal deletion mutant promote DNA renaturation.
