Characterization of RNase P RNA Activity

摘要: The principle task of the ubiquitous enzyme RNase P is generation mature tRNA 5'-ends by removing precursor sequences from primary transcripts (Trends Genet 19:561-569, 2003; Crit Rev Biochem Mol Biol 41:77-102, 2006; Trends Sci 31:333-341, 2006). In Bacteria, a ribonucleoprotein composed two essential subunits: catalytic RNA subunit (P RNA; 350-400 nt) and single small protein cofactor protein; ∼14 kDa). vitro, bacterial can catalyze maturation in absence at elevated concentrations mono- divalent cations (Cell 35:849-857, 1983). Thus, trans-acting multiple-turnover ribozyme.Here we provide protocols for 5'-endonucleolytic ptRNA cleavage RNAs any under single-turnover conditions ([E] >> [S]). Furthermore, outline concept that utilizes ribozyme to release interest with homogeneous 3'-OH ends via site-specific cleavage. Also, T7 transcription tRNAs clustered G residues 5'-end may result heterogeneities, which be avoided first transcribing 5'-precursor (ptRNA) followed RNA-catalyzed processing carrying 5'-monophosphate end. Finally, activity directly assayed using total extracts.