RNA sequence elements required for high affinity binding by the zinc finger domain of tristetraprolin: conformational changes coupled to the bipartite nature of Au-rich MRNA-destabilizing motifs.
Tristetraprolin (TTP) binds AU-rich elements (AREs) encoded within selected labile mRNAs and targets these transcripts for rapid cytoplasmic decay. RNA binding by TTP is mediated by an approximately 70-amino acid domain containing two tandemly arrayed CCCH zinc fingers. Here we show that a 73-amino acid peptide spanning the TTP zinc finger domain, denoted TTP73, forms a dynamic, equimolar RNA.peptide complex with a 13-nucleotide fragment of the ARE from tumor necrosis factor alpha mRNA, which includes small but significant contributions from ionic interactions. Association of TTP73 with high affinity RNA substrates is accompanied by a large negative change in heat capacity without substantial modification of RNA structure, consistent with conformational changes in the peptide moiety during RNA binding. Analyses using mutant ARE substrates indicate that two adenylate residues located 3-6 bases apart within a uridylate-rich sequence are sufficient for high affinity recognition by TTP73 (K(d) <20 nm), with optimal affinity observed for RNA substrates containing AUUUA or AUUUUA. Linkage of conformational changes and binding affinity to the presence and spacing of these adenylate residues provides a thermodynamic basis for the RNA substrate specificity of TTP.
Brewer
Brandy Y
BY
Department of Biochemistry and Molecular Biology, Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, 108 N. Greene Street, Baltimore, MD 21201, USA.
Malicka
Joanna
J
Blackshear
Perry J
PJ
Wilson
Gerald M
GM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016880:Anisotropy; D055592:Biophysical Phenomena; D001703:Biophysics; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D006358:Hot Temperature; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry*; D007477:Ions; D007700:Kinetics; D008954:Models, Biological; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D007477:Ions; D010455:Peptides; D012333:RNA, Messenger; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha; C497552:ZFP36 protein, human; D012313:RNA