The Escherichia coli unc transcription terminator enhances expression of uncC, encoding the epsilon subunit of F1-ATPase, from plasmids by stabilizing the transcript.
The effect of the unc transcription terminator on expression of uncC, encoding the epsilon subunit of Escherichia coli F1-ATPase, from plasmids was studied. The cloned sequence in pTK1 included the uncC ribosome binding site, the uncC structural gene, and the unc transcription terminator. The cloned region in pSD37 was similar, but lacked the unc transcription terminator. Transformants carrying pTK1 produced the epsilon subunit of F1-ATPase, encoded by uncC, in 10-fold greater abundance than transformants carrying pSD37. Northern blots revealed similar differences in the steady-state uncC mRNA levels. The half-life of the message transcribed from pTK1 was 90-100 seconds, while that from pSD37 was 25-30 seconds. These studies indicate the importance of message stabilization through features at the 3' end of the transcript in ensuring adequate production of epsilon. We have exploited this stabilization to develop a simple, efficient, and gentle method of purifying the overproduced epsilon subunit.
Patel
A M
AM
Department of Biochemistry, University of Western Ontario, London, Canada.
Dallmann
H G
HG
Skakoon
E N
EN
Kapala
T D
TD
Dunn
S D
SD
D015152:Blotting, Northern; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D012333:RNA, Messenger; D006180:Proton-Translocating ATPases