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A DNA-dependent RNA polymerase that transcribes vaccinia virus early genes was partially purified from virus cores by deoxycholate extraction and DEAE-cellulose column chromatography. Accurately initiated and terminated RNAs were synthesized by this enzyme in the presence of a linear duplex DNA template. Glycerol gradient sedimentation resolved the in vitro transcription system into two components: fraction I, a rapidly sedimenting RNA polymerase that initiated transcription at an early promoter but transcribed beyond the in vivo 3' terminus to yield a run-off transcript, and fraction II, a more slowly sedimenting fraction, itself devoid of RNA polymerase, that restored efficient termination when added back to fraction I. The termination factor was heat-labile, resistant to N-ethylmaleimide, and did not exhibit endonucleolytic activity on run-off transcripts. Factor-dependent termination required specific sequence information upstream of the site of termination. The vaccinia termination factor was purified extensively by column chromatography on DEAE-cellulose, heparin-agarose, phosphocellulose, and DNA-agarose, and by velocity sedimentation in a glycerol gradient. At each step, termination factor copurified with the vaccinia mRNA capping enzyme. The preparation was well over 90% pure with respect to the latter enzyme, suggesting that termination activity was tightly associated with, if not intrinsic to, the capping enzyme. Nonetheless, formation of the 5'-cap structure did not appear to be a prerequisite for termination.
Shuman et al. (Tue,) studied this question.