The regulatory protein MucR binds to a short DNA region located upstream of the mucR coding region in Rhizobium meliloti.
The Rhizobium meliloti MucR protein is known to regulate the biosynthesis of the two exopolysaccharides, succinoglycan and galactoglucan. The mucR gene was successfully overexpressed in Escherichia coli BL21 cells by heat shock induction using a two-plasmid system. Cell extracts of the production strain contained about 20% of a polypeptide of 17 kDa apparent molecular mass, corresponding to the size expected for MucR. As shown by an electrophoretic mobility shift assay, these extracts were active in the specific retardation of a 219-bp DNA fragment including 134-bp of the non-coding region upstream of the mucR gene. Primer extension analysis showed that this DNA fragment was located within the transcribed region upstream of the mucR gene. Competition experiments revealed that a 44-bp sequence present within the 134-bp upstream of the mucR gene contained the MucR binding site. Although binding of MucR to this site exhibited a moderate dissociation constant of Kd approximately 1.4 x 10(-7) M, the reaction was highly specific since fragments containing binding sites for the homologous Ros protein from Agrobacterium tumefaciens were not able to compete for MucR binding.
Bertram-Drogatz
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Universität Bielefeld, Fakultät fur Biologie, Lehrstuhl für Genetik,Germany.
Rüberg
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Becker
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Pühler
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D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D005809:Genes, Regulator / Q000235:genetics; D006706:Homeostasis / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012097:Repressor Proteins; D016962:Sinorhizobium meliloti / Q000235:genetics*; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C093770:MucR protein, Rhizobium meliloti; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors