Differential requirements for basic amino acids in transcription factor IIIA-5S gene interaction

Abstract

Basic amino acids Arg, Lys, and His in the Cys 2His 2 zinc fingers of transcription factor IIIA (TFIIIA) potentially have important roles in factor binding to the extended internal control region (ICR) of the 5S ribosomal gene. Conserved and non-conserved basic residues in the N-terminal fingers I, II, III and the more C-terminal fingers V and IX were analyzed by site-directed mutagenesis and DNase I protection in order to assess their individual requirement in the DNA-binding mechanism. In the DNA recognition helix of finger II, the conserved Arg at position 62 (N-terminal side of the first zinc-coordinating histidine) was changed to a Leu or Gln. Both the R62L and R62Q mutations inhibited Xenopus TFIIIA-dependent DNase I footprinting along the entire 5S gene ICR. When His-58 (non-conserved basic residue with DNA-binding potential in the same helical region) was changed to a Gln, the mutated protein was able to protect the ICR from DNase I digestion. Therefore, Arg-62 is individually required for TFIIIA binding over the entire ICR whereas His-58 is not. Fingers V and IX have conserved Arg residues in positions identical to Arg-62 in finger II (Arg-154 in finger V and Arg-271 in finger IX). When these residues were changed to Leu and Ile respectively, TFIIIA-dependent DNase I protection was observed along the entire 5S gene ICR. These results indicate differing DNA-binding mechanisms by the N-terminal fingers versus the C-terminal fingers at the level of individual amino acid-nucleotide interactions. In the N-terminal finger I, the conserved Lys at position 11 outside the recognition helix and a conserved hydrophobic Trp at position 28 within the helix were changed to an Ala and Ser respectively. The K11A change inhibited TFIIIA-dependent DNase I protection to a much greater extent than the W28S change.