Aminoacylation and micleoside modification of in vitro synthesised transfer RNA

Abstract

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, tRNA₁^Tyr (su₃⁺ and su₃⁻), tRNA₂^Gly su⁺A36, tRNA₃^Thr and tRNA₂^Tyr (carried by the respective ϕ80psu₃^+,− and λh80T phage DNA^2,3), 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 tRNA₁^Tyr su₃⁺ by crude S-30 E. coli extracts or a combination of partially purified fractions has been described^4–6. The formation of the primary transcription products of the tRNA^Tyr 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 synthesis^5,6. 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 (tRNA₁^Tyr, tRNA₂^Tyr, tRNA₃^Thr and tRNA₂^Gly) synthesised and processed in vitro, possess amino acid acceptance activity.