Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.
The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.
Green
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Cold Spring Harbor Laboratory, New York 11724.
Manche
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Mathews
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D000595:Amino Acid Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D012330:RNA, Double-Stranded / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D019892:eIF-2 Kinase
D012330:RNA, Double-Stranded; D012342:RNA, Small Nuclear; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase