Covalent enzyme-RNA complex: a tRNA modification that prevents a covalent enzyme interaction also prevents aminoacylation

Proc Natl Acad Sci U S A. 1985 Jan;82(2):339-42. doi: 10.1073/pnas.82.2.339.

Abstract

Previous work indicates that aminoacyl-tRNA synthetases make a transient covalent adduct with cognate tRNAs, through Michael addition of an enzyme nucleophile to the carbon-6 position of uridine 8. We report the selective reduction of the 5,6 double bond of 4-thiouridine at position 8 in Escherichia coli tyrosine tRNA, so as to prevent formation of the presumed covalent enzyme-nucleic acid adduct. The completely reduced tRNA molecules are inactivated for aminoacylation. With partial reduction, a mixed pool of active and inactive molecules is created and the degree of inactivation exactly matches the extent of 4-thiouridine reduction. The active molecules recovered from this mixed pool are specifically unaltered at position 8. The results are consistent with the view that the covalent enzyme-RNA adduct is an obligatory intermediate for aminoacylation of this tRNA.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Borates
  • Escherichia coli / genetics
  • Kinetics
  • Nucleic Acid Conformation*
  • RNA, Transfer / analysis*
  • RNA, Transfer, Amino Acyl / metabolism*
  • Thiouridine / metabolism

Substances

  • Borates
  • RNA, Transfer, Amino Acyl
  • Thiouridine
  • ammonium borate
  • RNA, Transfer