期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1993
卷号：90
期号：2
页码：472-476
DOI：10.1073/pnas.90.2.472
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We have developed an in vivo system to engender supercoiled "minicircle" DNA containing a single promoter by using the integrative recombination system of bacteriophage lambda. The resulting minicircle templates allow quantitative analysis of the stages of transcription initiation from a promoter, including synthesis of both full-length and aborted transcripts in the same reactions under physiological conditions. We have used such minicircle DNA templates to study in vitro transcription of the Escherichia coli gal promoter. The full-length transcripts from gal P1 and P2 promoters responded to cAMP-cAMP receptor protein in a manner identical to that observed in vivo. There is a 3.5-fold stimulation of P1 and almost total inhibition of P2 in the presence of cAMP. Thus, the unitary promoter system described here duplicates the in vivo physiology. In spite of the synthesis in equimolar amounts of full-length transcripts from P1 and P2 in the absence of cAMP in vitro, as in vivo, RNA polymerase encountered different rate-limiting steps of transcription initiation at the two promoters.
