Transcript RNA having trans-acting antitermination activity on the T7 transcription terminator

The efficiency of the phage T7 intrinsic terminator was determined in pulse-labeling in vitro transcription reactions. While the factor-independent terminator subcloned in pET3a showed consistently high (approximately 80%) efficiency, the efficiency of the same terminator in pGEM3ZT was initially approximately 60% but exponentially decreased to approximately 20%, although the 39-bp terminator, and its 73-bp upstream and 32-bp downstream sequences are identical in the two plasmids. When transcription product mixture of pGEM3ZT was added to an on-going reaction of pET3a, the terminator efficiency from pET3a was immediately reduced to approximately 40%. Furthermore, when the pGEM3ZT product mixture was subjected to the promoter-cleaving HinfI digestion and then phenol/chloroform extraction, the mixture still maintained the trans-acting antitermination activity. The results suggest that the trans-acting component(s) are RNA synthesized from pGEM3ZT.