Phage display of RNA binding zinc fingers from transcription factor IIIA.
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.
Friesen
W J
WJ
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Darby
M K
MK
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
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D012341:RNA, Ribosomal, 5S; D016601:RNA-Binding Proteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D004247:DNA