Selective RNA binding by a single CCCH zinc-binding domain from Nup475 (Tristetraprolin).
Regulation of gene expression takes place at several different levels and involves specific domains involved in specific protein-nucleic acid interactions. The protein Nup475 (also known as Tristetraprolin and TS11) binds to AU-rich sequence elements in certain mRNA molecules and favors the degradation of these mRNAs. The nucleic acid binding domain of Nup475 consists of two CCCH zinc-binding domains. A 36-amino acid peptide corresponding to the first of these CCCH domains has been synthesized and characterized. This peptide binds metal ions such as zinc(II) and cobalt(II) with affinities comparable to those of other authenticated zinc-binding domains. The zinc(II) complex of this peptide binds the RNA oligonucleotide UUUAUUU labeled with fluorescein on the 3'-end with an affinity of approximately 5 microM and discriminates against other sequences lacking the central A or the flanking U residues. These results demonstrate for the first time that a single CCCH domain is capable of binding single-stranded RNA with considerable affinity and selectivity. The combination of this well-behaved domain and the fluorescence-based binding assay sets the stage for more detailed structure-activity studies.
Michel
Sarah L J
SL
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Guerrerio
Anthony L
AL
Berg
Jeremy M
JM
D016880:Anisotropy; D001483:Base Sequence; D001665:Binding Sites; D003035:Cobalt / Q000378:metabolism; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D051816:Tristetraprolin; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D009843:Oligoribonucleotides; D010455:Peptides; D016601:RNA-Binding Proteins; D051816:Tristetraprolin; D003035:Cobalt