Purification and characterization of a transcription termination factor from vaccinia virions.
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.
D000227:Adenine Nucleotides / Q000494:pharmacology; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006358:Hot Temperature; D014157:Transcription Factors / Q000302:isolation & purification*; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014771:Virion / Q000201:enzymology* / Q000235:genetics
D000227:Adenine Nucleotides; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; D005033:Ethylmaleimide