DIFFERENTIAL REQUIREMENTS FOR BASIC-AMINO-ACIDS IN TRANSCRIPTION FACTOR IIIA-5S GENE INTERACTION

Basic amino acids Arg, Lys, and His in the Cys(2)His(2) zinc fingers o f 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 nonconserved 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 o rder to assess their individual requirement in the DNA-binding mechani sm. In the DNA recognition helix of finger II, the conserved Arg at po sition 62 (N-terminal side of the first zinc-coordinating histidine) w as changed to a Leu or Gin. Both the R62L and R62Q mutations inhibited Xenopus TFIIIA-dependent DNase I footprinting along the entire 5S gen e ICR. When His-58 (non-conserved basic residue with DNA-binding poten tial in the same helical region) was changed to a Gin, the mutated pro tein was able to protect the ICR from DNase I digestion, Therefore, Ar g-62 is individually required for TFIIIA binding over the entire ICR w hereas 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 Ar g-271 in finger IX). When these residues were changed to Leu and Ile r espectively, TFIIIA-dependent DNase I protection was observed along th e entire 5S gene ICR, These results indicate differing DNA-binding mec hanisms by the N-terminal fingers versus the C-terminal fingers at the level of individual amino acid-nucleotide interactions. In the N-term inal finger I, the conserved Lys at position II outside the recognitio n helix and a conserved hydrophobic Trp at position 28 within the heli x were changed to an Ala and Ser respectively. The K11A change inhibit ed TFIIIA-dependent DNase I protection to a much greater extent than t he W28S change. (C) 1998 Elsevier Science B.V. All rights reserved.