In Vitro Synthesis of tRNA: Identification and Processing of Primary Transcripts

Abstract

It is now generally established that mature tRNA molecules from both prokaryotic and eukaryotic sources are not direct transcription products but arise from the cleavage of longer precursor molecules (Schafer and Söll 1974; Altman 1975; Smith 1976). tRNA biosynthesis appears to involve posttranscriptional events, such as nucleolytic cleavage of precursors, nucleotide modifications at specific residues, and in some cases enzymatic terminal addition of CCA_OH Precursors to several RNAs specified by Escherichia coli and bacteriophage T4, which contain one or more tRNA sequences, have been isolated and characterized (Altman and Smith 1971; Barrell et al. 1974; Chang and Carbon 1975; Guthrie 1975; Sakano and Shimura 1975; Schedl et al. 1976; Ilgen et al. 1976). However, these tRNA precursors, which were shown to contain complete tRNA sequences plus extra nucleotides on the 5′ and 3′ ends and in the inter-tRNA spacer regions, seem to represent already partially cleaved products of an initially larger transcript, the nature of which remains unknown (Altman and Smith 1971; Barrell et al. 1974; Chang and Carbon 1975).

Transcription of tRNA genes in vitro by purified RNA polymerase has the advantage of producing completely unmodified tRNA precursors and makes it possible to study in detail the processing and modification of primary transcription products. The tRNA genes carried by the DNA of transducing bacteriophages φ80psu⁺3 (tRNA₁^Tyrsu⁺3 and su⁻3) and λh80dglyTsu⁺36 (tRNA₂^Tyr-tRNA₂^Glysu⁺36-tRNA₃^Thr) have been efficiently transcribed in vitro by purified E. coli RNA polymerase to produce high-molecular-weight tRNA precursors (Daniel et al. 1970; Grimberg and Daniel 1974). The...