Upstream curved sequences influence the initiation of transcription at the Escherichia coli galactose operon

• Published in: Journal of molecular biology Vol. 224; no. 2; pp. 293 - 306
• Main Authors: Lavigne, Marc, Herbert, Michel, Kolb, Annie, Buc, Henri
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 20.03.1992; Elsevier
• Subjects: Base Sequence; Biological and medical sciences; curved DNA sequences; cyclic AMP receptor protein; Deoxyribonuclease I - metabolism; Distamycins - pharmacology; DNA minor-groove binders; DNA, Bacterial; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; gal promoters; Galactose - genetics; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Kinetics; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; multiple kinetic pathways; Mutation; Netropsin - pharmacology; Nucleic Acid Conformation; Operon; Promoter Regions, Genetic - genetics; Regulatory Sequences, Nucleic Acid; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; curved DNA sequences; cyclic AMP receptor protein; multiple kinetic pathways; DNA minor-groove binders; gal promoters; Initiation complex; Upstream sequence; Molecular bending; Operon; Transcription promoter; Escherichia coli; Cyclic AMP; Bacteria; In vitro; Transcription initiation; Structure function relationship; Enterobacteriaceae
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/0022-2836(92)90995-V

The two overlapping promoters that control mRNA synthesis at the galactose operon contain three phased stretches of adenine residues, located around positions −84·5, −74 and −63, with respect to the start of the P1 promoter. As a result, the corresponding DNA sequence is bent, an anomaly that is relieved by the addition of small concentrations of drugs like distamycin A or netropsin. By abortive initiation assays performed on several DNA fragments derived from the wild-type promoter or from various mutants we show that the curved sequence increases the strength of the P1 promoter. In the absence of cyclic AMP (cAMP) and of the corresponding receptor protein (CRP), the upstream curved sequences enhance the rate of isomerization from the closed to the open complex at P1. This effect is abolished when distamycin A is bound in the bent region. In the presence of cAMP-CRP, a more drastic change is observed: activation of the gal P1 promoter takes place at a different formal step, depending whether the upstream curved sequence is present or not (enhancement of the rate of conversion from a closed to an open complex instead of an increase in the affinity of the enzyme during closed complex formation). These data, together with previous results obtained with other mutants of the gal control region, suggest that several closed complexes corresponding to different nucleoprotein arrangements are formed during open complex formation at gal P1, in the presence of CRP.