Differential requirements for basic amino acids in transcription factor IIIA-5S gene interaction.
Basic amino acids Arg, Lys, and His in the Cys2His2 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.
Hanas
J S
JS
Department of Biochemistry and Molecular Biology, University of Oklahoma College of Medicine, 940 Stanton Young Blvd., Oklahoma City, OK 73140, USA. jhanas@aardvark.ucs.uoknor.edu
Koelsch
G
G
Moreland
R
R
Wickham
J Q
JQ
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011485:Protein Binding; D012341:RNA, Ribosomal, 5S / Q000235:genetics*; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D006639:Histidine; D004247:DNA; D001120:Arginine