Highly efficient endonucleolytic cleavage of RNA by a Cys(2)His(2) zinc-finger peptide.
We have identified a 30-aa peptide that efficiently cleaves single-stranded RNA. The peptide sequence corresponds to a single zinc finger of the human male-associated ZFY protein; a transcription factor belonging to the Cys(2)His(2) family of zinc-finger proteins. RNA cleavage was observed only in the absence of zinc. Coordination with zinc resulted in complete loss of ribonuclease activity. The ribonuclease active structure was determined to be a homodimeric form of the peptide. Dimerization of the peptide occurred through a single intermolecular disulfide between two of the four cystines. The observed hydrolytic activity was single-stranded RNA-specific. Single-stranded DNA, double-stranded RNA and DNA, and 2'-methoxy-modified sequences were not degraded by the peptide. The peptide specifically cleaved pyrimidines within single-stranded RNA and the dinucleotide sequence 5'-pyr-A-3' was preferred. The RNA cleavage products consisted of a 3' phosphate and 5' hydroxyl. The initial rates of cleavage (V(0)) observed for the finger peptide were comparable to rates observed for human ribonucleases, and the catalytic rate (K(cat)) was comparable to rates observed for the group II intron rybozymes. The pH profile exhibited by the peptide is characteristic of general acid-base catalytic mechanisms observed with other ribonucleases. These observations raise interesting questions about the potential biological roles of zinc-finger proteins.
Lima
W F
WF
Department of Molecular and Structural Biology, Isis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA 92008, USA. wlima@isisph.com
Crooke
S T
ST
D000595:Amino Acid Sequence; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051741:Kruppel-Like Transcription Factors; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009843:Oligoribonucleotides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D012313:RNA / Q000737:chemistry*; D016415:Sequence Alignment; D013379:Substrate Specificity; D014157:Transcription Factors; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D009843:Oligoribonucleotides; D010455:Peptides; D014157:Transcription Factors; C054430:ZFY protein, human; D012313:RNA