In vitro processing of B. mori transfer RNA precursor molecules

Abstract

Ribonuclease P and 3′–5′ nuclease, two enzymatic activities necessary for tRNA synthesis in E. coli, are also found in the silkgland cells of Bombyx mori. B. mori subcellular extracts containing RNAase P activity can cleave the E. coli tRNA precursor molecule endonucleolytically at the same site as the E. coli enzyme, and will also cleave in vitro all E. coli tRNA precursors (pre-tRNAs) which the bacterial enzyme recognizes. B. mori RNAase P will not cleave two E. coli RNAase P substrates that are structurally unrelated to tRNA. Pre-tRNAs from B. mori contain extra 5′ and 3′ nucleotides as judged by RNA fingerprinting and 5′ terminal phosphate analysis. Crude silkgland extracts containing both RNAase P and 3′–5′ nuclease can remove the 5′ and 3′ extra nucleotides from B. mori pre-tRNAs, whereas purified fractions containing RNAase P remove only 5′ extra nucleotides. Only large silkworm pre-tRNAs were found to be susceptible to cleavage by B. mori RNAase P. This observation and sequence analysis of intermediates of in vitro processing reactions indicate a two-step process of pre-tRNA maturation in which extra 5′ nucleotides are first removed by RNAase P and extra 3′ nucleotides are then trimmed off by a 3′–5′ nuclease.