Contribution of the C-terminal tail of U1A RBD1 to RNA recognition and protein stability.
The N-terminal RNA binding domain (RBD1) of the human U1A protein interacts specifically with a short RNA hairpin containing the U1 snRNA stem/loop II sequence. Previous RNA binding studies have suggested that the C-terminal tail of RBD1 contributes to RNA recognition in addition to interactions on the beta-sheet surface of the protein. To evaluate the contributions of these C-terminal residues in RBD1 to RNA binding affinity and specificity, as well as to study the thermodynamic stability of RBDs, a number of RBD1 mutants with truncated tails, with single amino acid substitutions, and with both a truncation and an amino acid substitution, have been constructed. The thermodynamic stabilities of these mutants have been measured and compared by GdnHCI unfolding experiments. The RNA binding affinity and specificity of these mutant proteins have been assessed by measuring the binding of each protein to the wild-type RNA hairpin and to selected RNA mutants with nucleotide substitutions in the RNA loop. The results demonstrate first that, although the C-terminal tail of RBD1 makes significant contributions to RNA binding affinity, it is not required for RNA binding, and second, its contributions to binding specificity are mediated only through selected nucleotides in the RNA loop, for in the absence of the tail, the protein continues to use other nucleotides to discriminate among RNAs. In these truncated proteins, the secondary structure intrinsic to the C-terminal tail is absent, yet their affinity and discrimination for RNAs are not lost. Thus, a structured tail is not required for RNA recognition.
Zeng
Q
Q
Department of Biochemistry, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Hall
K B
KB
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000378:metabolism*
D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D012313:RNA