Specific binding of human dihydrofolate reductase protein to dihydrofolate reductase messenger RNA in vitro.
Dihydrofolate reductase (DHFR) is a critical enzyme in de novo purine and thymidylate biosynthesis. An RNA gel mobility shift assay was used to demonstrate a specific interaction between human recombinant DHFR protein and its corresponding DHFR mRNA. Incubation of DHFR protein with either its substrates, dihydrofolate or NADPH, or with an inhibitor, methotrexate, repressed its ability to interact with DHFR mRNA. An in vitro rabbit reticulocyte lysate translation system was used to show that the addition of exogenous human recombinant DHFR protein to in vitro translation reactions specifically inhibited DHFR mRNA translation. These studies suggest that the direct interaction between DHFR protein and its mRNA may be a mechanism for regulation of DHFR synthesis.
Chu
E
E
NCI-Navy Medical Oncology Branch, Division of Cancer Treatment, Bethesda, Maryland 20892.
Takimoto
C H
CH
Voeller
D
D
Grem
J L
JL
Allegra
C J
CJ
D001483:Base Sequence; D005492:Folic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005493:Folic Acid Antagonists; D005786:Gene Expression Regulation; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009249:NADP / Q000494:pharmacology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000187:drug effects / Q000378:metabolism*
D005493:Folic Acid Antagonists; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C010920:dihydrofolate; D009249:NADP; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate