Compilation and analysis of Escherichia coli promoter DNA sequences.

The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence.     

An analysis of the binding of repressor protein ModE to modABCD (molybdate transport) operator/promoter DNA of Escherichia coli.

Expression of the modABCD operon in Escherichia coli, which codes for a molybdate-specific transporter, is repressed by ModE in vivo in a molybdate-dependent fashion. In vitro DNase I-footprinting experiments identified three distinct regions of protection by ModE-molybdate on the modA operator/promoter DNA, GTTATATT (-15 to -8; region 1), GCCTACAT (-4 to +4; region 2), and GTTACAT (+8 to +14; region 3). Within the three regions of the protected DNA, a pentamer sequence, TAYAT (Y = C or T), can be identified. DNA-electrophoretic mobility experiments showed that the protected regions 1 and 2 are essential for binding of ModE-molybdate to DNA, whereas the protected region 3 increases the affinity of the DNA to the repressor. The stoichiometry of this interaction was found to be two ModE-molybdate per modA operator DNA. ModE-molybdate at 5 nM completely protected the modABCD operator/promoter DNA from DNase I-catalyzed hydrolysis, whereas ModE alone failed to protect the DNA even at 100 nM. The apparent K(d) for the interaction between the modA operator DNA and ModE-molybdate was 0.3 nM, and the K(d) increased to 8 nM in the absence of molybdate. Among the various oxyanions tested, only tungstate replaced molybdate in the repression of modA by ModE, but the affinity of ModE-tungstate for modABCD operator DNA was 6 times lower than with ModE-molybdate. A mutant ModE(T125I) protein, which repressed modA-lac even in the absence of molybdate, protected the same region of modA operator DNA in the absence of molybdate. The apparent K(d) for the interaction between modA operator DNA and ModE(T125I) was 3 nM in the presence of molybdate and 4 nM without molybdate. The binding of molybdate to ModE resulted in a decrease in fluorescence emission, indicating a conformational change of the protein upon molybdate binding. The fluorescence emission spectra of mutant ModE proteins, ModE(T125I) and ModE(Q216*), were unaffected by molybdate. The molybdate-independent mutant ModE proteins apparently mimic in its conformation the native ModE-molybdate complex, which binds to a DNA sequence motif of TATAT-7bp-TAYAT.     

Tandem DNA-bound cAMP-CRP complexes are required for transcriptional repression of the deoP2 promoter by the CytR repressor in Escherichia coli

We have studied the deoP2 promoter in Escherichia coli to define features important for its interaction with the CytR repressor. As is characteristic for CytR-regulated promoters, deoP2 encodes tandem binding sites for the activating complex cAMP-CRP. One of these sites, CRP-1, overlaps the -35 region, and is sufficient for activation; the second site, CRP-2, centred around -93, is indispensable for repression. Here we demonstrate, by means of in vivo titration, that CytR interaction with deoP2 depends not only on CRP-2, but also on CRP-1 and the length and possibly the sequence separating these two sites. Also, point mutations in either CRP site reduce or abolish CytR titration; however, no co-operativity is observed in the interaction of CytR with the two CRP binding sites. Furthermore, the reduction in CytR titration parallels the reduction in binding of cAMP-CRP to the mutated CRP sites in vitro. These observations are not easily explained by current models for the action of prokaryotic repressors; instead we favour a model in which the interaction of CytR with deoP2 depends on the presence of tandem DNA-bound cAMP-CRP complexes.     

Mutational analysis of the Escherichia coli serB promoter region reveals transcriptional linkage to a downstream gene.

Genes encoding proteins with unrelated functions can be cotranscribed, and this may be used by cells to coordinate different metabolic pathways during growth. We describe a gene, designated sms, which is downstream from the serine biosynthetic gene serB in Escherichia coli but does not appear to be involved in amino acid (aa) biosynthesis. The sms gene is 1380 bp long. The Sms product migrates at 55 kDa on sodium dodecyl sulfate(SDS)-polyacrylamide gels and has a M(r) of 49472 (460 aa residues) calculated from the nucleotide sequence. The deduced Sms aa sequence shares regions of similarity with two ATP-dependent proteases, Lon and RecA, and contains two motifs: a C-x(2)-C-x(n)-C-x(2)-C motif, which is found in some nucleic acid binding proteins, and an ATP/GTP binding site motif. Insertional inactivation of sms led to increased sensitivity to the alkylating agent methylmethane sulfonate, but not to a requirement for serine or other metabolites. Several promoter mutations were isolated and characterized, which suggest that serB has a typical promoter recognized by sigma 70. After the serB coding sequence there is a 48-bp region with no obvious promoter sequence preceding the sms translation start codon. Analyses using sms'-lacZ fusions cloned downstream from wild-type and mutant serB promoters showed that sms is cotranscribed with serB.     

Identification of the DNA binding surface of H-NS protein from Escherichia coli by heteronuclear NMR spectroscopy.

The DNA binding domain of H-NS protein was studied with various N-terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D- and 3D-NMR spectroscopies. It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H-NS falls in the range from 1/6 to 1/25 for H-NS(60-137), H-NS(70-137) and H-NS(80-137), whereas it was much weaker for H-NS(91-137). Thus, the DNA binding domain was defined to be the region from residue A80 to the C-terminus. Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60-R93 in mutant protein H-NS(60-137) forms a long stretch of disordered, flexible chain, and also showed that the structure of the C-terminal region (residues A95-Q137) in mutant H-NS(60-137) was nearly identical to that of H-NS(91-137). 1H and 15N chemical shift perturbations induced by complex formation of H-NS(60-137) with an oligonucleotide duplex 14-mer demonstrated that two loop regions, i.e. residues A80-K96 and T110-A117, play an essential role in DNA binding.