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Nucleic Acids Research, 2003, Vol. 31, No. 16 4882-4887
© 2003 Oxford University Press

Role of cysteine amino acid residues on the RNA binding activity of human thymidylate synthase

Xiukun Lin, Jun Liu, Frank Maley1 and Edward Chu*

Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine and VA Connecticut Healthcare System, New Haven, CT 06520, USA and 1 Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201, USA

*To whom correspondence should be addressed at: VA Connecticut Healthcare System, Cancer Center, 111-D, 950 Campbell Avenue, West Haven, CT 06516, USA. Tel: +1 203 937 3421; Fax: +1 203 937 3803; Email: chueyale{at}yahoo.com

The role of cysteine sulfhydryl residues on the RNA binding activity of human thymidylate synthase (TS) was investigated by mutating each cysteine residue on human TS to a corresponding alanine residue. Enzymatic activities of TS:C43A and TS:C210A mutant proteins were nearly identical to wild-type TS, while TS:C180A and TS:C199A mutants expressed >80% of wild-type enzyme activity. In contrast, TS:C195A was completely inactive. Mutant proteins, TS:C195A, TS:C199A and TS:C210A, retained RNA binding activity to nearly the same degree as wild-type human TS. RNA binding activity of TS:C43A was reduced by 30% when compared to wild-type TS, while TS:C180A was completely devoid of RNA binding activity. In vitro translation studies confirmed that mutant proteins TS:C43A, TS:C195A, TS:C199A and TS:C210A, significantly repressed human TS mRNA translation, while TS:C180A was unable to do so. To confirm the in vivo significance of the cysteine sulfhydryl residue, mutant proteins TS:C180A and TS:C195A were each expressed in human colon cancer HCT-C18:TS(–) cells that expressed a functionally inactive TS. A recombinant luciferase reporter gene under the control of a TS-response element was co-transfected into these same cells, and luciferase activity increased in the presence of the TS:C195A mutant TS protein to a level similar to that observed upon expression of wild-type TS protein. In contrast, luciferase activity remained unchanged in cells expressing the TS:C180A mutant protein. Taken together, these findings identify Cys-180 as a critical residue for the in vitro and in vivo translational regulatory effects of human TS.


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C. Du, R. Niu, E. Chu, P. Zhang, and X. Lin
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J. Biochem., May 1, 2006; 139(5): 913 - 920.
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