Partial Purification and Characterization of RNase P from Arabidopsis Thaliana Tissue

摘要

A distinct ribonuclease P (RNase P) activity that cleaves leader sequences from precursor tRNA (ptRNA) molecules to give mature 5, ends has been isolated from Arabidopsis thaliana tissue. The RNase P activity was isolated by ammonium sulfate precipitation of a tissue homogenate and further purified by anion exchange chromatography. The isolated activity is capable of processing tobacco ptRNA(GIY) and cyanobacterial ptRNA(Gln) substrates in vitro. The two tested substrates are processed both with and without a terminal 3 CCA sequence which, when present, increases processing by the catalytic subunit of Escherichia coli RNase P. The isolated activity was shown to be sensitive to micrococcal nuclease and proteinase K indicating that a ribonucleoprotein is essential for catalytic activity. Database mining of the Arabidopsis genome revealed a possible homology for the Rpp38 subunit of human RNase P holoenzyme. Western blot analysis of the isolated RNase P activity revealed cross-reactivity with human Rpp3 8 antibody; specifically, a 1 9-kDa Arabidopsis protein is identified by the Rpp38 antibody. These results strongly suggest that A. thaliana RNase P is a ribonucloprotein complex and is similar to that isolated from other eukaryotic systems.