A regulatory cascade in the induction of rhaBAD.
The RhaS and RhaR regulatory proteins are encoded in the Escherichia coli L-rhamnose gene cluster. We used complementation analysis and DNA mobility shift assays to show that RhaR is not the direct activator of the L-rhamnose catabolic operon, rhaBAD. An in-frame deletion of rhaS (rhaS-rhaR+) eliminated expression from the rhaBAD promoter, pBAD, while overexpression of rhaS greatly speeded the normally slow induction of transcription from pBAD. Expression from pBAD in a coupled transcription-translation assay was only detected when rhaS+ DNA was added to allow synthesis of RhaS protein. RhaS thus appears to be the direct L-rhamnose-specific activator of rhaBAD expression. Deletion mapping located the binding site for the L-rhamnose-specific regulator to a region overlapping position -70 relative to the rhaBAD transcription start site. Deletion mapping and DNA mobility shift assays located a CRP binding site just upstream from the binding site for the L-rhamnose-specific regulator. Quantitative primer extension analysis showed that induction of both the rhaBAD and rhaSR messages was unusually slow, requiring 40 to 50 minutes to reach a steady-state level. Induction of rhaBAD apparently involves a regulatory cascade in which RhaR first induces rhaSR expression, then RhaS accumulates and induces rhaBAD expression.
Egan
S M
SM
Biology Department, Johns Hopkins University, Baltimore, MD 21218.
Schleif
R F
RF
D001426:Bacterial Proteins / Q000502:physiology; D001483:Base Sequence; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic; D012210:Rhamnose / Q000378:metabolism*; D015534:Trans-Activators; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C062863:RhaR protein, E coli; C084095:RhaS protein, E coli; D015534:Trans-Activators; D014157:Transcription Factors; D012210:Rhamnose