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.
Lee
J T
JT
Department of Life Science, Korea Advanced Institute of Science and Technology, Taejon.
Kang
C
C
D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D010957:Plasmids; D012367:RNA, Viral / Q000235:genetics*; D013604:T-Phages / Q000235:genetics*; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins
D012367:RNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases