Comparative utilization of pseudouridine triphosphate and uridine triphosphate by ribonucleic acid polymerase.

The relatively large amounts of pseudouridine in that fraction of the ribonucleic acid concerned with the transfer of activated amino acids to ribosomes for peptide bond formation (l-4) suggests that this unusual base may play an essential role in this reaction, and that a specific mechanism exists for its incorporation into interribonucleotide linkage. To obtain information on the nature of this specificity, we have prepared pseudouridine triphosphate (5) and studied its incorporation into RNA (6) by a preparation of RNA polymerase obtained from nuclear extracts of HeLa cells (7). These experiments indicated that fi UTP can substitute with comparable efficiency for UTP in the synthesis of RNA from the four nucleoside triphosphates. When labeled in its innermost phosphate,l the Pa2 of $ UTP32 is carried into interribonucleotide linkage as the pseudouridyl nucleotide without prior conversion or transfer to a uridyl compound. The labeling pattern of the 2’(3’)-nucleoside monophosphates released on alkaline hydrolysis of the synthesized radioactive RNA (expressed as the ratio of radioactivity associated with each particular 2’(3’)-nucleoside monophosphate compared with 2’(3’)-AMP taken as 1.00) is identical with that found with UTPs2. The addition of fi UTP to the four required nucleoside triphosphates (including UTP) did not stimulate the rate of RNA synthesis. However, study of the labeling pattern produced in the presence of both fi UTP and UTP, one labeled with P32 and the other not, revealed pronounced changes in the ratios of alkali released P32-2’(3’)-nucleoside monophosphates. These changes were dependent on the relative concentrations of the isomeric nucleoside triphosphates and were in opposite directions depending on which of the isomers was used as the radioactive compound. The data could be interpreted as indicating a preference on the part of the incorporating pseudouridylic acid to be linked through its 5’.phosphate group to the 3’-hydroxyl group of purine nucleosides, whereas uridylic acid was linked preferentially to pyrimidine nucleosides. The fact that no changes in the labeling pattern with CTP3” or ATPs2 were found when both unlabeled isomers were present at different relative concentrations is against the selection of a new DNA template on addition of $ UTP and UTP.