Molecular biology of vertebrate transcription factor IIIA: cloning and characterization of TFIIIA from channel catfish oocytes

Abstract

TFIIIA regulates 5S rRNA synthesis and is the prototype of the Cys 2His 2 superfamily of zinc finger proteins. Because the TFIIIA aa sequence is highly diverged, elucidating species variation in this factor will yield insights into how zinc fingers bind DNA and how this protein regulates RNAPIII transcription. This study reports the identification, cloning and functional divergence of oocyte TFIIIA from the channel catfish. Catfish oocyte TFIIIA was identified by its association with 5S rRNA in immature ovarian tissue, its molecular weight, and by peptide sequence similarities with Xenopus TFIIIA. The cDNA for this factor was cloned by degenerate PCR and found to code for nine Cys 2His 2 zinc fingers and a C-terminal tail; only about 40% aa sequence identity was observed with Xenopus TFIIIA. The N-terminal region of catfish TFIIIA contains the oocyte-specific initiating Met amino acid and accompanying conserved residues found in amphibian TFIIIAs but not found in yeast or human TFIIIAs. Catfish TFIIIA lacks the conserved transcription activation domain in its C-terminal tail found in amphibian and human TFIIIA. Catfish TFIIIA was able to bind the catfish and Xenopus 5S RNA genes but did not efficiently promote 5S gene transcription in a rodent RNAPIII transcription system, as did Xenopus TFIIIA. Amino acid conservation in catfish, amphibian, and human TFIIIA zinc fingers allows deduction of possible finger recognition helix alignments along the conserved 5S gene ICRs. For the three N-terminal fingers, this leads to deduction of a compact polypeptide structure with conserved basic residues contacting conserved G nts in the 5S gene C box.