Synthetic curved DNA sequences can act as transcriptional activators in Escherichia coli.
Can a transcriptional activator known to bend DNA be functionally replaced by a sequence-directed bend in Escherichia coli? To investigate this question, a partially truncated promoter was used, deleted of its -35 region and of its CRP binding site, leaving only two Pribnow boxes as functional elements. Synthetic and naturally occurring curved DNA sequences introduced upstream from these elements could restore transcription at either one of the two natural starts. Some of these hybrid promoters turned out to be more efficient than the CRP activated wild-type gal promoter in vivo. Control experiments performed with very similar sequences devoid of any curvature produced weak promoters only. Minimal changes in the location of the centre of curvature or perturbation in the amount of curvature strongly affected the level of expression. No significant stimulation of transcription could be detected in vitro. Furthermore, both gal P1 and P2 starts could be activated in vivo but also in vitro via a properly positioned CRP binding site. This partial analogy suggests that bending induced by the cAMP-CRP complex upon binding to its site may be biologically relevant to the mechanism of transcriptional activation.
Bracco
L
L
Institut Pasteur, Unité de Physicochimie des Macromolécules Biologiques (CNRS: URA 1149), Paris, France.
Kotlarz
D
D
Kolb
A
A
Diekmann
S
S
Buc
H
H
D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004278:DNA, Superhelical / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005838:Genotype; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D015534:Trans-Activators; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000235:genetics
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D001616:beta-Galactosidase