PUL ID

PUL0068

PubMed

8277949, Mol Gen Genet. 1994 Jan;242(1):90-9. doi: 10.1007/BF00277352.

Characterization method

enzyme activity assay, electrophoretic mobility shift assay

Genomic accession number

AM886293.1

Nucelotide position range

23902-29914

Substrate

raffinose

Loci

rafRABD

Species

Escherichia coli/562

Degradation or Biosynthesis

degradation

Gene Name

Locus Tag

Protein ID

Gene Position

GenBank Contig Range

EC Number

RafR IPF_354 CAP07693.1 10 - 1021 (+) AM886293.1:23912-24923 -
RafA IPF_353 CAP07694.1 1116 - 3243 (+) AM886293.1:25018-27145 -
RafB IPF_349 CAP07695.1 3305 - 4583 (+) AM886293.1:27207-28485 -
RafD IPF_347 CAP07696.1 4582 - 6013 (+) AM886293.1:28484-29915 -

Cluster number

1

Gene name

Gene position

Gene type

Found by CGCFinder?

RafR 11 - 1021 (+) TF: DBD-Pfam|LacI,DBD-SUPERFAMILY|0036955 No
RafA 1117 - 3243 (+) CAZyme: GH36 Yes
RafB 3306 - 4583 (+) TC: gnl|TC-DB|P16552|2.A.1.5.2 Yes
RafD 4583 - 6013 (+) CAZyme: GH32 Yes

PUL ID

PUL0068

PubMed

8277949, Mol Gen Genet. 1994 Jan;242(1):90-9. doi: 10.1007/BF00277352.

Title

Role of two operators in regulating the plasmid-borne raf operon of Escherichia coli.

Author

Muiznieks I, Schmitt R

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, O1 and O2, that flank the -35 sequence of the raf promoter, PA. In vitro, O1 and O2 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 configuration or mutant O1 or O2. When either one of the two operators was inactivated by site-directed mutagenesis, both O1 and O2 exhibited the same affinity for repressor and the same sensitivity to arrest of repressor binding by the natural inducer, melibiose. However, in the native O1O2 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 O2 alone exerted 45% and O1 alone 6% repression of rafA transcription. The differential effects of O1 versus O2 on transcription (despite matching affinities of O1 and O2 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 O1 serves to enhance this effect.