The Biosynthesis of Pseudouridine in Ribonucleic Acids of Escherichia coli

The utilization of pseudouridine-2-14C by a uracil-requiring mutant of Escherichia coli is described. Cells were grown in the presence of labeled pseudouridine (Ψ uridine) and suboptimal amounts of uracil. During one doubling time a small amount of Ψ uridine was taken up, and radioactivity appeared in DNA and RNA. Small amounts of pseudouridine and its mono-, di-, and triphosphates were isolated from the metabolic pool. The high specific activities of these compounds indicated that little dilution of fed Ψ uridine had occurred in the pool. In RNA, label appeared mainly in the pyrimidines, with Ψ uridine being the most highly labeled. Appearance of label in uridylic and cytidylic acids is considered to result from a limited ability of the organism to degrade Ψ uridine. The specific activities of Ψ uridine isolated from the various RNA fractions and from the common tetranucleotide T-Ψ-C-G of tRNA were 0.2 to 2% that of ΨTP of the metabolic pool. An inverse relationship was observed among the RNA fractions between the content of Ψ uridine and its specific activity. Thus, the specific activity of Ψ uridine in 16 S rRNA > 23 S RNA > tRNA > T-Ψ-C-G. We conclude that Ψ uridine is not incorporated as such into positions in RNA where it normally occurs, but that non-specific incorporation can occur.