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Interaction at a distance between multiple operators controls the adjacent, divergently transcribed glpTQ-glpACB
T J Larson1, J S Cantwell, A T van Loo-Bhattacharya
1Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308.
Abstract:
The glp regulon of Escherichia coli encodes the proteins required for utilization of sn-glycerol 3-phosphate and its precursors. Transcription of the divergently transcribed glpTQ and glpACB operons is initiated at sites separated by 132 base pairs (bp) of DNA. These operons are controlled negatively by glp repressor and positively by the cAMP-cAMP receptor protein (CRP) complex. The locations of the binding sites for the glp repressor and for cAMP.CRP in the control regions of these operons were determined by DNase I footprinting. Binding of the glp repressor protected the region -32 to -51 (OT) in the glpTQ promoter, which was also the binding site for cAMP.CRP. Four repressor binding sites (-41 to -60 (OA1), -9 to -28 (OA2), +12 to -8 (OA3), and +52 to +33 (OA4)) and two cAMP.CRP binding sites (+11 to -11 and -30 to -51) were found in the glpACB promoter region. Comparison of the sequences of the repressor binding sites found in the glpTQ-glpACB control region with those operators previously described in the glpD operon allowed formulation of a consensus operator sequence which was the palindrome 5'-WATGTTCGWTAWC-GAACATW-3' (W is A or T). The role of each operator was assessed by measuring repression in constructs where individual operators were altered by site-directed mutagenesis. Alteration of OT did not significantly decrease repression of either operon. Each of the glpACB operators contributed to repression of both operons. These results suggest involvement of glpACB operator(s) in control of glpTQ expression perhaps via formation of a repression loop. Evidence supporting this hypothesis was obtained by measuring the degree of repression of the glpTQ promoter in constructs containing 6- or 10-bp insertions between the glpTQ and glpACB operators. A 6-bp insertion located within OA2 or between OT and OA1 eliminated repression of the glpTQ promoter, whereas significant repression was maintained in the case of a 10-bp insertion within OA2.