Characteristics of the interaction of a synthetic human tristetraprolin tandem zinc finger peptide with AU-rich element-containing RNA substrates.
Tristetraprolin (TTP) and its two known mammalian family members are tandem CCCH zinc finger proteins that can bind to AU-rich elements (AREs) in cellular mRNAs and destabilize those transcripts, apparently by initiating their deadenylation. Previous studies have shown that the approximately 70-amino acid tandem zinc finger domain of TTP is required and sufficient for RNA binding, and that the integrity of both zinc fingers is also required. However, little is known about the kinetics or structure of the peptide-RNA interaction, in part because of difficulties in obtaining soluble recombinant protein or peptides. We characterized the binding of a synthetic 73-amino acid peptide from human TTP to the tumor necrosis factor (TNF) ARE by gel mobility shift analyses and fluorescence anisotropy experiments. Both types of studies yielded a peptide-RNA dissociation constant of approximately 10 nM. Surprisingly, we found that the "footprint" from the TNF ARE required for peptide binding was only approximately 9 bases and that two molecules of peptide could bind to probes containing as little as 19 bases. An identical recombinant peptide exhibited gel shift characteristics similar to those of the synthetic peptide. NMR analysis of the 15N-labeled recombinant peptide suggested that its first zinc finger was structured in solution but that the second was not. The titration of oligonucleotides representing 17, 13, and even 9 bases of the TNF ARE caused an essentially identical, dramatic shift of existing resonances, and the appearance of new resonances in the peptide spectra, so that all amino acids could be assigned. These data suggest that this TTP peptide-RNA complex is structured in solution and might be amenable to NMR structure determination.
Blackshear
Perry J
PJ
Laboratory of Signal Transduction, Research Triangle Park, North Carolina 27709, USA. black009@niehs.nih.gov
Lai
Wi S
WS
Kennington
Elizabeth A
EA
Brewer
Gary
G
Wilson
Gerald M
GM
Guan
Xiaoju
X
Zhou
Pei
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D012313:RNA / Q000378:metabolism*; D051816:Tristetraprolin; D016335:Zinc Fingers
D017931:DNA Primers; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D051816:Tristetraprolin; C497552:ZFP36 protein, human; D012313:RNA