URIDINE INSERTION INTO PREEDITED MESSENGER-RNA BY A MITOCHONDRIAL EXTRACT FROM LEISHMANIA-TARENTOLAE - STEREOCHEMICAL EVIDENCE FOR THE ENZYME CASCADE MODEL

An RNA editing-like internal uridine (U) incorporation activity (G. C. Frech, N. Bakalara, L. Simpson, and A. M. Simpson, EMBO J. 14:178-187 , 1995) and a S'-terminal U addition activity (N. Bakalara, A. M. Simp son, and L. Simpson, J. Biol. Chem. 261:15679-18686, 1989) have been p reviously described by using a mitochondrial extract from Leishmania t arentolae. Chiral phosphorothioates were used to investigate the stere oconfiguration requirements and the stereochemical course of these nuc leotidyl transfer reactions. The extract utilizes (S-p)-alpha-S-UTP fo r both 3' and internal U incorporation into substrate RNA. The interna l as well as the 3' incorporation of (S-p)-alpha-S-UTP proceeds via in version of the stereoconfiguration. Furthermore, internal U incorporat ion does not occur at sites containing thiophosphodiesters of the R(p) configuration. Our results are compatible with an enzyme cascade mode l for this in vitro U insertion activity involving sequential endonucl ease and uridylyl transferase directly from UTP and RNA ligase steps a nd are incompatible with models involving the transfer of U residues f rom the 3' ends of guide RNAs.