In vitro transcription and isolation of a polycistronic RNA product of the T4 tRNA operon

Abstract

ALTHOUGH the expression of bacteriophage T4 genome has been extensively studied¹, no direct relationship has been established between specific genes and their immediate transcripts. Escherichia coli RNA polymerase transcribes in vitro a T4 DNA region of about 50 genes which tend to cluster according to function² and are expressed early after bateriophage infection. One of these clusters is the tRNA region coding for eight tRNA species and two stable RNA species of unnown function which appear after bacteriophage infection^3,4. Deletion mutants lacking different sequences in this region have been isolated and well characterised⁵ (Fig. 1). T4-specific tRNAs can be produced in vitro by transcribing T4 DNA with E. coli RNA polymerase and processing the unfractionated product with E. coli extracts⁶. Although the temporal order of appearance of T4 tRNA species in the in vitro transcription-processing system suggested that the tRNA genes may be organised as a single transcription unit⁷, the formation of a long polycistronic precursor was not demonstrated. Here we describe the fractionation of RNA transcripts produced on T4 DNA by E. coli RNA polymerase into several high molecular weight RNA species. Using as templates DNA from T4 mutants containing deletions in the tRNA gene region we identified a polycistronic precursor of T4 tRNAs among the primary transcription products. This precursor was isolated and processed into mature size tRNA molecules.