Characterization of the gcv control region from Escherichia coli.
We constructed a set of deletions upstream of the gcv promoter and analyzed the effects of the deletions on expression of a gcvT-lacZ gene fusion. A deletion that ends at position -313 upstream of the transcription initiation site (+1) results in reduced levels of gcvT-lacZ expression, but the fusion is still inducible by glycine and repressible by purines. A deletion that ends at position -169 results in loss of both GcvA- and Lrp-mediated activation of the gcvT-lacZ fusion. The endpoints of delta -313 and delta -169 also define a site that down-regulates gcvT-lacZ expression two- to threefold. A deletion that ends at position -89 upstream from the transcription initiation site still shows PurR-mediated repression, suggesting that PurR-mediated repression is not by direct interference with the GcvA- and Lrp-mediated regulatory mechanism(s). Gel mobility shift assays and DNase I footprinting showed that Lrp protein binds to multiple sites upstream of the gcv promoter, from about bp -92 to bp -229. The results suggest that the gcv regulatory region is complex, with numerous cis-acting sites that are required for normal gcv expression.
Stauffer
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Department of Microbiology, University of Iowa, Iowa City 52242.
Stauffer
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D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D050960:Aminomethyltransferase; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015971:Gene Expression Regulation, Enzymologic; D005998:Glycine / Q000378:metabolism; D019877:Hydroxymethyl and Formyl Transferases; D051756:Leucine-Responsive Regulatory Protein; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion; D014157:Transcription Factors; D014166:Transferases / Q000096:biosynthesis / Q000235:genetics*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C497278:Lrp protein, E coli; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C064525:glycine cleavage system; D051756:Leucine-Responsive Regulatory Protein; D000594:Amino Acid Oxidoreductases; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; D050960:Aminomethyltransferase; D005998:Glycine