Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition
AUTOR(ES)
Tai, Ningwen
FONTE
Oxford University Press
RESUMO
Previous studies have shown that human dihydrofolate reductase (DHFR) acts as an RNA-binding protein, in which it binds to its own mRNA and, in so doing, results in translational repression. In this study, we used RNA gel mobility shift and nitrocellulose filter-binding assays to further investigate the specificity of the interaction between human DHFR protein and human DHFR mRNA. Site-directed mutagenesis was used to identify the critical amino acid residues on DHFR protein required for RNA recognition. Human His-Tag DHFR protein specifically binds to human DHFR mRNA, while unrelated proteins including thymidylate synthase, p53 and glutathione-S-transferase were unable to form a ribonucleoprotein complex with DHFR mRNA. The Cys6 residue is essential for RNA recognition, as mutation at this amino acid with either an alanine (C6A) or serine (C6S) residue almost completely abrogated RNA-binding activity. Neither one of the cysteine mutant proteins was able to repress the in vitro translation of human DHFR mRNA. Mutations at amino acids Ile7, Arg28 and Phe34, significantly reduced RNA-binding activity. An RNA footprinting analysis identified three different RNA sequences, bound to DHFR protein, ranging in size from 16 to 45 nt, while a UV cross-linking analysis isolated an ∼16 nt RNA sequence bound to DHFR. These studies begin to identify the critical amino acid residues on human DHFR that mediate RNA binding either through forming direct contact points with RNA or through maintaining the protein in an optimal structure that allows for the critical RNA-binding domain to be accessible.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=137126Documentos Relacionados
- Identification of Amino Acid Residues in the Caenorhabditis elegans POU Protein UNC-86 That Mediate UNC-86–MEC-3–DNA Ternary Complex Formation
- Delayed processing of dihydrofolate reductase heterogeneous nuclear RNA in amino acid-starved mouse fibroblasts.
- Amino-Acid Sequence of Dihydrofolate Reductase from a Methotrexate-resistant Mutant of Streptococcus faecium and Identification of Methionine Residues at the Inhibitor Binding Site
- Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.
- Identification of amino acid residues critical for infection with ecotropic murine leukemia retrovirus.