Purification and characterization of a transcription termination factor from vaccinia virions.

Abstract

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.

• Authors: S Shuman, S S Broyles, B Moss
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 262 Issue 25 Pg. 12372-80 (Sep 05 1987) ISSN: 0021-9258 [Print] United States
• PMID: 3624264 (Publication Type: Journal Article)

Chemical References

• Adenine Nucleotides
• Transcription Factors
• DNA-Directed RNA Polymerases
• Ethylmaleimide

Topics

• Adenine Nucleotides (pharmacology)
• Base Sequence
• DNA-Directed RNA Polymerases (isolation & purification, metabolism)
• Ethylmaleimide (pharmacology)
• Hot Temperature
• Transcription Factors (isolation & purification)
• Transcription, Genetic
• Vaccinia virus (enzymology, genetics)
• Virion (enzymology, genetics)