INTERACTION OF DEACYLATED YEAST PHENYLALANINE TRANSFER-RNA WITH ESCHERICHIA-COLI RIBOSOMES - ROLE OF MODIFIED NUCLEOTIDE IN CODON ANTICODONINTERACTION

Derivatives of yeast phenylalanine tRNA (tRNA(GmAAY)(Phe)) modified at position 37 (tRNA(GAAA)(Phe)) and at both the anticodon and position 37 (tRNA(GCAG)(Phe)) were obtained by enzymatic replacement of the ant icodon loop. A quantitative study of the interaction of different type s of deacylated tRNA(Phe) (tRNA(GmAAY)(Phe), tRNA(GAAA)(Phe), tRNA(GCA G)(Phe), and tRNA(-Y)(Phe)) with the P site of the 70S ribosome-poly(U ) complex was carried out at different Mg2+ concentrations and tempera tures. Upon replacement of the Y base with adenosine, the interaction enthalpy decreases from 39 to 24 kcal/mole, whereas upon its removal t he enthalpy drops to 16 kcal/mole. Replacement of the second base of t he anticodon, adenosine, by cytosine leads to a decrease in the intera ction enthalpy to 6 kcal/mole, which is characteristic of the interact ion of tRNA with the P site in the absence of poly(U). In the absence of a template the affinity of tRNA(-Y)(Phe) to the P Site of 70S ribos omes is fivefold lower than that for tRNA(GmAAY)(Phe) and tRNA(GCAG)(P he). Thus, the modified nucleotide not only stabilizes codon-anticodon interaction by stacking with the codon-anticodon bases, but also redu ces the free energy of binding by interacting with the hydrohobic cent er of the P site.