During the last decade, the regulation of the proU operon has been extensively studied, in particular, with regard to H-NS (21). However, it has recently been demonstrated that the mechanism of proU repression by H-NS cannot be explained solely by the binding of the regulator to the promoter region (28). 2). These results suggest that the region downstream of the +1 transcriptional start site plays a crucial role in the positive control by H-NS of flagellum biosynthesis in vivo. In the present study, we attempted to determine more precisely the role of H-NS and cAMP-CAP on flagellar gene expression and, in particular, on the flhDC master operon. Similarly, plasmid pDIA559 was constructed by PCR amplification of the fliCpromoter with the primers 5′-GGGATCCGTAAAACGAATACCGGG-3′ and 5′-CCCAAGCTTGGTATTAATGACTTGTGCC-3′. A preferential binding of H-NS to the flhDCpromoter was observed when the concentration of H-NS reached 2 μM (Fig. Kolb A, Busby S, Herbert M, Kotlarz D, Buc H. Biochemistry. The electrophoretic mobility of this DNA-protein complex was further retarded in the presence of H-NS. Strains carrying thehns-1001 (7) andcrp::Sm (29a) mutations were constructed by P1 transduction with phage P1vir as previously described (39). Recently, interactions between H-NS and various regulators have been demonstrated. Nucleotide substitutions identified in the flhDC promoter of a cfs strain are indicated by arrowheads. The ability of CAP to distinguish between restriction fragments and genomic DNA, shown by the difference in binding cooperativity, suggests the existence of previously unsuspected DNA sequences or structures that modulate its binding cooperativity. Examining Primer extension analysis was performed with RNA extracted from a wild-type strain carrying plasmid pDIA551. In vitro transcription experiments performed with H-NS showed that this regulator represses in vitro expression of the master operon (Fig. Around 50 distinct AKAPs have been discovered so far and are found to be responsible for colocalizing PKA to its specific substrates such as ion channels and cytoskeletal elements. In this case, transcription occurs only at a low level. Reconstitution of the [4Fe-4S] cluster in FNR and demonstration of the aerobic-anaerobic transcription switch in vitro. By continuing you agree to the use of cookies. CAP helps the efficient binding of RNA polymerase to the promoter. Kumar S. Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli. These results are in agreement with those obtained by footprinting experiments (see below). Taking into account the ionic strength of the cell cytosol (∼ 200 mM), the physiological cAMP concentrations (below 10 µM) and the CRP dissociation constant value for cAMP binding (∼ 10 µM), the biologically relevant CRP species are the apo-CRP and the CAP:(cAMP)1 complex.
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