Identification of an RNA binding site for human thymidylate synthase.
Previous studies from this laboratory have shown that human TS mRNA translation is regulated by its protein product in a negative autoregulatory manner. In this paper, we identify an RNA binding site for TS protein located within the first 188 nt of TS RNA. A 36-nt RNA sequence contained within this 188-nt fragment, corresponding to nt 75-110 and including the translational initiation site, binds TS protein with an affinity similar to that of both the full-length and the 188-nt TS RNA sequences. Variant RNAs with either a deletion or a mutation at the translational initiation region are unable to compete for TS protein binding. UV crosslinking studies reveal that an RNA fragment of approximately 36 nt is protected from RNase T1 digestion by TS protein binding. A second TS protein-binding site is localized within the protein-coding region corresponding to nt 434-634. These findings demonstrate a specific interaction between human TS protein and its TS RNA and identify an RNA binding site that includes the translational initiation site.
Chu
E
E
National Cancer Institute-Navy Medical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Voeller
D
D
Koeller
D M
DM
Drake
J C
JC
Takimoto
C H
CH
Maley
G F
GF
Maley
F
F
Allegra
C J
CJ
D001483:Base Sequence; D004252:DNA Mutational Analysis; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D014466:Ultraviolet Rays
D012333:RNA, Messenger; D013940:Thymidylate Synthase