Effects of 5-fluorouracil substitution on the RNA conformation and in vitro translation of thymidylate synthase messenger RNA

C. H. Takimoto, D. B. Voeller, J. M. Strong, L. Anderson, E. Chu, C. J. Allegra

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

In vitro transcribed thymidylate synthase (TS) mRNA which is 100% substituted with 5-fluorouracil (FUra) was analyzed for changes in mRNA secondary structure, for alterations in translational efficiency, and for evidence of translational miscoding in vitro. FUra substitution in TS mRNA results in an altered migration pattern in non-denaturing RNA gels and in decreased hyperchromicity in RNA melting temperature studies, consistent with a change in mRNA secondary structure. However, no change in the translational efficiency of FUra-substituted TS mRNA is seen compared to control TS mRNA in either rabbit reticulocyte lysate or wheat germ extract in vitro translation systems. Analysis of the in vitro translation product of FUra-substituted TS mRNA by Western immunoblotting, isoelectric focusing, 5-fluoro-2'- deoxyuridine 5'-monophosphate binding, and TS catalytic activity experiments shows no difference compared to control TS mRNA. We conclude that the in vitro translation products of FUra-substituted and control TS mRNA are identical. Our findings do not support the hypothesis that changes in the mRNA template are responsible for the RNA-directed cytotoxicity of FUra.

Original languageEnglish (US)
Pages (from-to)21438-21442
Number of pages5
JournalJournal of Biological Chemistry
Volume268
Issue number28
StatePublished - 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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