CHARACTERIZATION AND PARTIAL-PURIFICATION OF 2 PRE-TRANSFER-RNA 5'-PROCESSING ACTIVITIES FROM DAUCUS-CARROTA (CARROT) SUSPENSION CELLS

Two distinct RNase P-like activities which cleave leader sequences from pre-tRNA molecules to give mature 5' ends have been identified in carrot suspension-culture cells. An Escherichia coli pre-tRNA(Phe) and a tobacco pretRNA(Tyr) were transcribed in vitro then used as substrates for processing reactions in a cell-free extract. The pretRNA(Tyr) transcript was used to establish optimal salt and divalent cation requirements for processing. Kinetic experiments were then carried out on both substrates to determine if 5' and 3' processing were ordered. Primer extension analysis of processing intermediates and stable products verified that an ammonium sulfate fraction of the extract was indeed capable of accurately processing the 5' ends of both pre-tRNAs. Subsequent fractionation of the 5' end-processing activity by chromatography on phosphocellulose revealed two distinct activities, eluting at 0.1 and 0.5 M KCl, when assayed with the tobacco pretRNA(Tyr) substrate. When the same fractions were assayed with the E. coli pre-tRNA(Phe), only the 0.1 M KCl fraction exhibited activity. Both of the active fractions display sensitivity to micrococcal nuclease (MN) and proteinase K indicating each is a ribonucleoprotein, a result not seen with other plant RNase Ps. Subsequent FPLC fractionation of the two activities using Mono Q and Mono S columns demonstrated that the two activities could be further distinguished on the basis of their chromatographic behavior.