A Preferential Role for lysyl-tRNA4 in the Synthesis of Diadenosine 5',5'''-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA Synthetase Complex From Rat Liver

Biochemistry. 1987 Mar 24;26(6):1586-91. doi: 10.1021/bi00380a015.

Abstract

The synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits. This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase. We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture. Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys). No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast. Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine. None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture. Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex. This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations. The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.

Publication types

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

MeSH terms

  • Adenine Nucleotides / biosynthesis*
  • Amino Acyl-tRNA Synthetases / metabolism*
  • Animals
  • Arginine-tRNA Ligase / metabolism*
  • Dinucleoside Phosphates*
  • Kinetics
  • Liver / enzymology*
  • Lysine-tRNA Ligase / metabolism*
  • Multienzyme Complexes / metabolism*
  • RNA, Transfer, Amino Acyl / metabolism*
  • Rats
  • Zinc / analysis

Substances

  • Adenine Nucleotides
  • Dinucleoside Phosphates
  • Multienzyme Complexes
  • RNA, Transfer, Amino Acyl
  • diadenosine tetraphosphate
  • Amino Acyl-tRNA Synthetases
  • Arginine-tRNA Ligase
  • Lysine-tRNA Ligase
  • Zinc