| 摘要 |
<p>A system to construct supercoiled 'minicircle' DNA containing a single promoter by the use of the integrative recombination system of bacteriophage lambda is described. The supercoiled DNA minicircles are templates which carry a single promoter. Such minicircles serve as templates to study the steps of transcription initiation from a unitary promoter and to synthesize both full length transcripts and aborted transcripts under physiological conditions in the absence of other contamining promoter activities. The invention is directed to in vitro transcription of promoters, such as the E. coli gal promoter, using such minicircle DNA templates. The full-length transcripts from g^_a^_l^_ P^_1 and P^_2 promoters responded to cAMP CRP in a manner identical to that observed in vivo. In addition, an in vitro transcription assay, using minicircle DNA containing the g^_a^_l^_ promoter with l^_a^_c^_ operators, was employed to elucidate the molecular mechanism of repression. Lac repressors, which associate into a tetramer and form a DNA loop, repressed transcription from P^_1 and P^_2, while a non-looping Lac repressor mutant failed to show normal repression of both of the g^_a^_l^_ promoters.</p> |
