title: Phage display of RNA binding zinc fingers from transcription factor IIIA.
abstract: Zinc fingers in transcription factor IIIA (TFIIIA) contribute differentially to RNA and DNA binding affinity. We investigated whether the same putative alpha-helix amino acids in TFIIIA zinc fingers are essential for both RNA and DNA binding. In published structures, zinc fingers make DNA base contacts through amino acids -1, +2, +3, and +6 of the recognition helix. Alanine substitution at these four positions were made in TFIIIA RNA binding zinc fingers, tz4-7 and DNA binding zinc fingers, tz1-3. Substitution in zinc fingers 4 or 6 of tz4-7 reduced RNA affinity 77- and 38-fold, respectively, whereas substitution in zinc fingers 5 or 7 had little effect. DNA binding affinity of tz1-3 was eliminated by alanine substitution in any one zinc finger. We determined which amino acids supported RNA binding by phage display of a library of zinc finger 4 mutants. Lysine at helix position -1 of zinc finger 4 was conserved in all selected tz4-7 fusions. Point mutation of Lys-1 to alanine in zinc finger 4 reduced tz4-7 RNA affinity 30-fold. We propose that RNA binding by TFIIIA shows similarity to DNA binding in the use of the recognition helix. Helix positions -1 and +2 may have particular significance for RNA binding.
authors.affiliation: Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: W J
authors.initials: WJ
authors.lastname: Friesen
authors.forename: M K
authors.initials: MK
authors.lastname: Darby
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D012341:RNA, Ribosomal, 5S / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D012341:RNA, Ribosomal, 5S; D016601:RNA-Binding Proteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D004247:DNA