Nuclease digestion of synthetase x tRNA complexes

Phenylalanyl-tRNA and seryl-tRNA synthetase protect strongly though not completely their cognate tRNAs against nuclease attack, as had been shown previously. In an investigation of the mechanism of protection it was demonstrated that the low susceptibility of phenylalanyl-tRNA-synthetase x tRNA-Phe complexes to nucleases is due to free tRNA present in equilibrium with synthetase. The equilibrium can be shifted by an excess of synthetase or by dilution of the complex. It therefore appears that synthetase competes with the nuclease for free tRNA. Degradation of the complex is low, however, because under the conditions of partial digestion the synthetase has a greater affinity for the tRNA than does the nuclease. Fragmented tRNAs, as they are formed during partial nuclease digestion, bind to synthetase to different degrees. tRNA-Phe with a lesion in the dihydrouridine loop binds very poorly whereas a nick in the anticodon loop reduces the strength of binding to a much lesser extent. In a systematic study of the stoichiometry of protection it was confirmed that under standard conditions one phenylalanyl-tRNA synthetase protects one tRNA-Phe and one seryl-tRNA synthetase two tRNA-Ser molecules against nuclease attack. Under certain conditions, however, (concentration of the complex higher than 10 mu-M, or alternately in buffers of low ionic strength) it is observed that phenylalanyl-tRNA synthetase binds up to 1.6 molecules tRNA-Phe. In the serine system, these special conditions do not affect the binding properties of seryl-tRNA synthetase.