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Nucleobase Amino Acids Incorporated into the HIV‐1 Nucleocapsid Protein Increased the Binding Affinity and Specificity for a Hairpin RNA
Author(s) -
Takahashi Tsuyoshi,
Ueno Akihiko,
Mihara Hisakazu
Publication year - 2002
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/1439-7633(20020603)3:6<543::aid-cbic543>3.0.co;2-r
Subject(s) - rna , zinc finger , nucleobase , amino acid , chemistry , binding site , biochemistry , binding selectivity , nucleic acid , nucleic acid structure , cytosine , stereochemistry , dna , gene , transcription factor
L ‐ α ‐amino acids with a nucleobase in the side chain (nucleobase amino acids; NBAs) were used to enhance the function of RNA‐binding proteins that recognize structured RNA. These NBAs were utilized in the three‐dimensional structure of the protein to enhance RNA binding affinity and specificity as a result of selective recognition of NBAs by RNA bases. NBA units were incorporated at various positions into the HIV‐1 nucleocapsid protein NCp7 (residues 1–55), which contains two CCHC‐type (Cys‐X 2 ‐Cys‐X 4 ‐His‐X 4 ‐Cys‐type; X=an amino acid residue) zinc knuckle domains. The binding ability was evaluated by using the stem‐loop (SL)3 region of HIV‐1 Ψ‐RNA. Visible light absorption measurements revealed that two zinc ions bound strongly and quantitatively to the NBA‐NCp7 molecule and to the wild‐type NCp7 protein. This result indicates that the incorporation of NBA units composed of L ‐ α ‐amino acids did not influence the formation of the specific structure of NCp7. Binding analysis with fluorescein‐labeled SL3 RNA revealed that incorporation of NBA units into the NCp7 protein at appropriate positions increased its RNA binding affinity and specificity. An NBA‐NCp7 protein that possessed cytosine and guanine NBA units at positions 13 and 46, respectively, showed a binding affinity for SL3 RNA ninefold higher than that of wild‐type NCp7 as a result of the specific and cooperative interaction of the NBA units with RNA bases. These results clearly demonstrate that inclusion of NBA units in the three‐dimensional structure of an RNA‐binding protein is a useful strategy for enhancing the function of the protein.