Efficient Control of raf Gene Expression by CAP and Two Raf Repressors that Bend DNA in Opposite Directions
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I. Muiznieks
Abstract
The plasmid-borne raf operon of Escherichia coli encodes proteins involved in the uptake and utilisation of the trisaccharide raffinose. The operon is subject to dual regulation; to negative control by the binding of RafR repressor to twin operators, O1 and O2, and to positive control by the cAMP-binding protein, CAP. We have identified the CAP binding site (CBS) as a 22 bp palindromic sequence with incomplete dyad symmetry by deletion analysis, DNaseI footprinting and electrophoretic mobility shift assays (EMSA) of CAP-DNA complexes. The CBS is centred 60.5 bp upstream of the transcription start point and partially overlaps O1. In vivo, CAP increases rafA (α-galactosidase) gene expression up to 50-fold. The 28 bp spacing between the centres of CBS and the—35 box is essential, since insertions of 4, 8, 12 or 16 bp completely eliminated rafA gene expression. In vitro binding studies revealed that the CBS, O1 and O2 sites, can be simultaneously occupied by their cognate proteins. However, no cooperativity between binding of CAP and RafR was detected. EMSA with circularly permuted DNA fragments demonstrated that CAP and RafR proteins bend raf promoter (rafP) DNA by 75° ± 5° and 95° ± 5°, respectively, in opposite directions. Among sugar catabolic operons, the compact arrangement of three protein-binding sites, a CBS and two operators bounding the—35 promoter box, is unique and provides a sensitive and highly efficient device for transcriptional control.
Copyright © 1999 by Walter de Gruyter GmbH & Co. KG
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Artikel in diesem Heft
- To Our Authors, Readers and Subscribers. Biological Chemistry 1999 - Increased Page Number and Online Accessibility
- Proto-Oncogenes, Unlike Harmless Genes, Tend to Be Dispersed in the Human Genome: Selection Against Out-of-Register Recombination?
- The Human H2A and H2B Histone Gene Complement
- Efficient Control of raf Gene Expression by CAP and Two Raf Repressors that Bend DNA in Opposite Directions
- Molecular Models of Acidic PePtides From Pea Bud Chromatin And Seminal Plasma. Divalent Cations-Mediated Interaction With Dna
- Cytidine Triphosphate Synthase Activity and mRNA Expression in Normal Human Blood Cells
- Studies with Lysine N6-Hydroxylase. Effect of a Mutation in the Assumed FAD Binding Site on Coenzyme Affinities and on Lysine Hydroxylating Activity
- The Recombinant Thermosome from the Hyperthermophilic Archaeon Methanopyrus kandleri: In Vitro Analysis of Its Chaperone Activity
- Not More than Three Tissue Kallikreins Identified from Organs of the Guinea Pig
- Activation of Protein C by Arginine-Specific Cysteine Proteinases (Gingipains-R) from Porphyromonas gingivalis
- Alteration of Mosaic Methylation of the Repeat Unit of the Human Ribosomal RNA Genes in Lung Cancer
- Degradation of the Cyclic AMP Antagonist Prostaglandylinositol Cyclic Phosphate (Cyclic PIP) by Dephosphorylation
- Homo-Dimeric Spherulin 3a: A Single-Domain Member of the bg-Crystallin Superfamily
- cDNA Sequencing of Guinea Pig a2-HS Glycoprotein, Its Expression in Various Tissues and Acute Phase Expression
- Phage Display Selection of P1 Mutants of BPTI Directed against Five Different Serine Proteinases
