RNA polymerase idling and clearance in gal promoters: use of supercoiled minicircle DNA template made in vivo.

Abstract:

: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.

authors

Choy HE,Adhya S

doi

10.1073/pnas.90.2.472

subject

Has Abstract

pub_date

1993-01-15 00:00:00

pages

472-6

issue

2

eissn

0027-8424

issn

1091-6490

journal_volume

90

pub_type

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