Expression vector pT7:TKII for the synthesis of authentic biologically active RNA encoding vaccinia virus thymidine...

A transcription vector, pT7: TKII, was constructed by a novel application of the polymerase chain reaction. Chimeric oligodeoxynucleotides were used to direct the synthesis of a DNA fragment which consisted of a truncated bacteriophage T7 promoter element fused to the vaccinia virus (VV) thymidine kinase gene (tk). This fragment was cloned into a pUC118 plasmid and sequenced to ensure no mutations had occurred during its synthesis. When linearized at the 3' end of the VV tk gene at the BamHI site located in the polylinker region of the vector, pT7:TKII was efficiently transcribed by T7 RNA polymerase into a 595 nucleotide transcript whose 5' end was identical to that found on authentic nascent VV tk mRNA. When translated in a rabbit reticulocyte lysate system, the synthetic VV tk RNA was shown to be biologically active in that it directed the synthesis of a 20-kDa protein which assembled into an enzymatically active 80-kDa tetrameric complex which was indistinguishable from VV thymidine kinase (TK) enzyme isolated from VV-infected cells. The pT7:TKII vector provides a powerful approach with which: (i) to investigate the translational and posttranslational regulation of the VV tk gene; (ii) to use directed genetics to identify potential cis-acting regulatory sequences or structures present within the VV tk RNA; and (iii) to apply protein engineering procedures to identify the catalytic, allosteric and subunit interactive domains of the VV TK enzyme. As an example, the translational effects of adding a m7G cap structure to the pT7:TKII-derived VV tk RNA are presented.