The Function of Pseudouridylic Acid in Transfer Ribonucleic Acid

Abstract Inhibition of the nonenzymatic binding of phenylalanyl transfer RNA to polyuridylic acid-coded ribosomes at 20 mm magnesium was used as an assay to determine whether the tetranucleotide ribothymidylyl-pseudouridylyl-cytidylyl-guanosine 3'-phosphate possessed any specific ability to bind to ribosomes. Inhibition of binding at the peptidyl site was studied by adding tetracycline to block aminoacyl site binding activity, and aminoacyl site activity was examined in the presence of excess deacylated tRNA to suppress peptidyl site binding. Deacylated tRNA was bound competitively with aminoacyl-tRNA at the peptidyl site, but was without effect at the aminoacyl site. At both of these sites, T-ψ-C-Gp was more inhibitory than any other oligonucleotide tested. At the peptidyl site, T-ψ-C-Gp was 10 times more active than A-A-A-Gp, and 3 times more active than (A, U, C)Gp. At the aminoacyl site, T-ψ-C-Gp was again 3 times more active than (A, U, C)Gp and 20 times more active than A-A-Gp or (A,C)Gp. Alkaline hydrolysis destroyed the inhibitory activity and cyanoethylation of the pseudouridine residue with acrylonitrile decreased the activity to 50% although a control treated with propionitrile was fully active. Inhibitory activity was resistant to boiling. Inhibition by T-ψ-C-Gp was not competitive with free phenylalanyl-tRNA nor affected by ribosome concentration. However, T-ψ-C-Gp does act at the ribosomal level since ammonium chloride-washed particles were 4 times more sensitive to T-ψ-C-Gp inhibition, and aging of these particles further increased their sensitivity. Variation of the polyuridylic acid or Mg++ concentration was without effect. Since T-ψ-C-Gp was able to block aminoacyl-tRNA binding sites on ribosomes more effectively than other similar nucleotides this sequence probably represents part of the normal ribosome binding site in tRNA. The failure to compete with unbound aminoacyl-tRNA and the effect of ammonium chloride washing on the ribosomes suggests that the actual reaction mechanism is complex.