MUIZNIEKS, I and SCHMITT, R (1994) ROLE OF 2 OPERATORS IN REGULATING THE PLASMID-BORNE RAF OPERON OF ESCHERICHIA-COLI. MOLECULAR & GENERAL GENETICS, 242 (1). pp. 90-99. ISSN 0026-8925,
Full text not available from this repository.Abstract
The plasmid-borne raf operon encodes functions required for the inducible uptake and utilization of raffinose in Escherichia coli K12. The expression of three structural genes for alpha-galactosidase (rafA), Raf permease (rafB) and sucrose hydrolase (rafD) is negatively controlled by the binding of RafR repressor (rafR) to two operator sites, O-1, and O-2, that flank the -35 sequence of the raf promoter, P-A. In vitro, O-1, and O-2, are occupied on increasing the concentration of RafR, without detectable preference for one site or the other or any indication of cooperative binding. Nucleotide substitutions at positions 3, 4 or 5 in an operator half-site prevented repressor binding, supporting a model that postulates specific interactions of these base pairs with the recognition helix of RafR. To study the role of each operator site, we have compared by gel shift analysis the binding of purified RafR repressor to DNA fragments containing the original O1O2,0, configuration or mutant O-1 or O-2. When either one of the two operators was inactivated by site-directed mutagenesis, both O-1 and O-2 exhibited the same affinity for repressor and the same sensitivity to arrest of repressor binding by the natural inducer, melibiose. However, in the native O-1,O-2 configuration, simultaneous binding of RafR to both operators was sterically hindered, leading to a 13-fold decrease in the intrinsic affinity of an operator site for repressor, once the other site had been occupied. To assess the role of each operator in vivo, rafA was used as a reporter gene. A 1200-fold repression (100%) was exerted by RafR binding to the native O1O2 configuration, whereas O-2 alone exerted 45% and O-1 alone 6% repression of rafA transcription. The differential effects of O-1 versus O-2 on transcription (despite matching affinities of O-1 and O-2 for repressor) suggest that positioning of the O-2-repressor complex between the -35 and -10 signals is crucial for transcription control and that repressor binding to the upstream O-1 serves to enhance this effect.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PROTEIN-DNA RECOGNITION; LAC REPRESSOR; NUCLEOTIDE-SEQUENCES; GEL-ELECTROPHORESIS; PROMOTER; BINDING; RAFFINOSE; GENES; TRANSCRIPTION; ELEMENTS; RAF OPERON; DUAL OPERATOR CONTROL; REPRESSOR-OPERATOR BINDING; SITE-DIRECTED MUTAGENESIS; GEL RETARDATION |
| Depositing User: | Dr. Gernot Deinzer |
| Last Modified: | 19 Oct 2022 08:41 |
| URI: | https://pred.uni-regensburg.de/id/eprint/53584 |
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