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RECENT evidence suggests that the structural genes of some transfer RNA molecules are clustered in groups of two or three on the Escherichia coli chromosome1–5. The arrangement of tRNA and spacer sequences for two such tRNA clusters have been described6–8. The pattern of transcription of tRNA genes in clusters is not known although the formation of larger precursors in tRNA biosynthesis has been reported9,10. In vitro transcription of tRNA1Tyr gene carried by ϕ80psu3+ phage DNA by purified RNA polymerase was shown to result in the formation of pre-tRNATyr molecules bigger in size than mature tRNA11. In the present communication we describe the transcription of a tRNA gene cluster (tRNA2Gly(su+36), tRNA2Tyr, tRNA3Thr) carried by the DNA of transducing phage λh80cI857S−t68dglyTsu+36tyrTthrT (abbreviated λh80T) recently isolated by Squires et al.5. In vitro transcription of this phage DNA by purified RNA polymerase is shown to produce pre-tRNA molecules of a molecular size consistent with the model of a polycistronic precursor.

TRANSCRIPTION of transfer RNA (tRNA) genes in vitro by purified RNA polymerase has the advantage of producing completely unmodified tRNA precursors which provide a substrate for the study of the processes and modifications leading to the formation of mature tRNA molecules. We have shown1 that the tRNA genes, tRNA1Tyr (su3+ and su3−), tRNA2Gly su+A36, tRNA3Thr and tRNA2Tyr (carried by the respective ϕ80psu3+,− and λh80T phage DNA2,3), were transcribed in vitro by Escherichia coli RNA polymerase into polycistronic tRNA precursors. These precursors were cleaved on incubation with a supernatant fraction from E. coli, yielding mature size tRNA molecules1. The transcription in vitro of an active tRNA1Tyr su3+ by crude S-30 E. coli extracts or a combination of partially purified fractions has been described4–6. The formation of the primary transcription products of the tRNATyr gene was not, however, clearly demonstrated and it was concluded that an additional E. coli extract fraction would be required to enable purified RNA polymerase to function in tRNA synthesis5,6. We report here evidence indicating that the in vitro transcription of ϕ80psu3+,− or λh80T DNA by purified E. coli RNA polymerase alone produces tRNA precursor molecules which are not only processed to form mature-size tRNA1 but are also recognised by specific modifying enzymes. In addition the tRNA molecules (tRNA1Tyr, tRNA2Tyr, tRNA3Thr and tRNA2Gly) synthesised and processed in vitro, possess amino acid acceptance activity.

Two Escherichia coli tRNA gene clusters, tRNA1Tyr (su3+ and su3-) and tRNA2Tyr, tRNA2Gly (su+36), tRNA3Thr, were transcribed in a purified in vitro system. Evidence indicates that the adjacent tRNA genes are transcribed together as a common precursor of large size, which, on incubation with crude cell extracts, yields mature tRNA molecules.

In bacteriophage T4 DNA, transcription units recognized in vitro by host RNA polymerase consist of promotor-proximal 'immediate early' (IE) genes and promotor-distal 'delayed early' (DE) genes separated from each other by rho-dependent transcription terminators. In vivo, the transition from IE to DE transcription requires phage-specific protein synthesis and can be prevented by chloramphenicol (CAM). Most of the information about IE/DE transition has been obtained by hybridizaton analyses of mixtures of RNA species synthesized simultaneously on several T4 transcription units (for review see ref. 3). A useful model for the study of T4 gene expression at the level of primary transcripts and individual gene products is provided by the T4 tRNA operon, a cluster of genes coding for eight T4-specific transfer RNAs and two stable RNAs (species 1 and 2) of unknown function (Fig. 1). The 10 genes of the tRNA operon are arranged in two subclusters (I and II) with a promotor located about 1 kilobase pair upstream. The primary transcripts and the final gene products of this region have been identified and isolated. Moreover, this genetic region was recently cloned and a part of it sequenced. We describe here the expression of T4 tRNA genes in vivo and in vitro in terms of the IE/DE concept and demonstrate that the two subclusters of the tRNA operon are subject to different modes of control.

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 infection3,4. 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.