ISSN:
1617-4623
Keywords:
raf operon
;
Dual operator control
;
Repressor-operator binding
;
Site-directed mutagenesis
;
Gel retardation
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
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 α-galactosidase (rafA), Raf permease (rajB) and sucrose hydrolase (rafD) is negatively controlled by the binding of RafR repressor (rafR) to two operator sites, O1 and O2, that flank the − 35 sequence of the raf promoter, PA. In vitro, O1 and 02 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 0102 configuration or mutant O1 or 02. When either one of the two operators was inactivated by site-directed mutagenesis, both 01 and 02 exhibited the same affinity for repressor and the same sensitivity to arrest of repressor binding by the natural inducer, melibiose. However, in the native 0102 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 02 alone exerted 45% and 01 alone 6% repression of rafA transcription. The differential effects of 01 versus 02 on transcription (despite matching affinities of 01 and 02 for repressor) suggest that positioning of the O2-repressor complex between the — 35 and —10 signals is crucial for transcription control and that repressor binding to the upstream 01 serves to enhance this effect.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00277352
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