DNA-binding specificity of the Lon protease α-domain from Brevibacillus thermoruber WR-249

Lon protease has been well studied in many aspects; however, the DNA-binding specificity of Lon in prokaryotes has not been clearly identified. Here we examined the DNA-binding activity of Lon protease α-domains from Brevibacillus thermoruber (Bt), Bacillus subtilis (Bs), and Escherichia coli (Ec). MALDI-TOF mass spectroscopy showed that the α-domain from Bt-Lon binds to the duplex nucleotide sequence 5′-CTGTTAGCGGGC-3′ (ms1) and protected it from DNase I digestion. Surface plasmon resonance showed that the Bt-Lon α-domain binds with ms1 double-stranded DNA tighter than Bs- and Ec-Lon α-domains, whereas the Bt-Lon α-domain has dramatically lower affinity for double-stranded DNA with 0 and 50% identity to the ms1 binding sequence. Our results indicated that Bt-Lon α-domain plays a critical role with ms1 sequence in the DNA-binding specificity.     

In Vitro Interaction of Nuclear Proteins with the Promoter of Soybean Heat Shock Gene Gmhsp17.5E

Proteins present in crude nuclear extracts of soybean (Glycine max) plumules were shown to bind in vitro to the 5′ flanking sequences of the soybean heat shock gene Gmhsp17.5E. The specificity of binding activity present in extracts from both control (28°C) and heat shocked (40°C) tissues was demonstrated by reciprocal competition experiments using gel mobility retardation assays. Footprinting experiments using DNase I with crude nuclear extracts indicated that a continuous stretch of 5′ flanking sequences extending from −40 to −153 was protected from digestion in vitro. Nuclear proteins that were partially purified by heparin agarose chromatography were shown to bind specific TATA-proximal sequences containing the heat shock consensus elements (HSEs) (−73 to −49; −107 to −84) and AT-rich motifs (−119 to −153). Other binding sites within AT-rich sequences (−906 to −888, −868 to 863, −859 to 853, and −841 to −830), distal HSE elements (−568 to −532) and a TATA/dyad (−234 to −207) were also identified by DNase I footprinting of TATA-distal probes. DNA binding activities specific for the HSE and AT-rich sequences were present in nuclear extracts from both control and heat shocked tissues. Both types of binding activity were increased after heat shock treatment; HSE binding increased from 1.8- to 2.7-fold, and binding to AT-rich sequences showed an increase from 1.3- to 1.7-fold.     

Binding of ArsR,the repressor of the Staphylococcus xylosus (pSX267) arsenic resistance operon to a sequence with dyad symmetry within the ars promoter

arsR, the first gene of the Staphylococcus xylosus (pSX267) arsenic/antimonite resistance (rs) operon encodes a negative regulatory protein, ArsR, which mediates inducibility of the resistances by arsenic and antimony compounds. ArsR, which has no obvious DNA-binding motif in its primary structure, was purified from an ArsR-overproducing Escherichia coli strain and identified as a DNA-binding protein by its behaviour in gel mobility shift assays. ArsR had a specific affinity for a 312 by DNA restriction fragment carrying the ars promoter; the minimum sequence complexed by ArsR was a 75 by polymerase chain reaction (PCR) fragment, which mainly comprised the ?35 and ?10 regions of the promoter. The effect of inducers on the DNA-binding activity of ArsR was examined by in vitro induction assays; only arsenite inhibited DNA-binding of the repressor. DNase I footprinting revealed two protected regions within the promoter region, spanning 23 and 9 nucleotides, respectively. Furthermore, a new cleavage site for DNase I between the protected regions was made accessible by binding of the repressor. The footprints cover a region of three inverted repeats located between the ?35 and ?10 motifs of the ars promoter. By high resolution footprinting with the hydroxy radical, five sites of close contact between the protein and DNA were identified.     

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity.     

Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

DNase I hypersensitive (DHS) sites are important for understanding cis regulation of gene expression. However, existing methods for detecting DHS sites in small numbers of cells can lead to ambiguous results. Here we describe a simple new method, in which DNA fragments with ends generated by DNase I digestion are isolated and used as templates for two PCR reactions. In the first PCR, primers are derived from sequences up- and down-stream of the DHS site. If the DHS site exists in the cells, the first PCR will not produce PCR products due to the cuts of the templates by DNase I between the primer sequences. In the second PCR, one primer is derived from sequence outside the DHS site and the other from the adaptor. This will produce a smear of PCR products of different sizes due to cuts by DNase I at different positions at the DHS site. With this design, we detected a DHS site at the CD4 gene in two CD4 T cell populations using as few as 2×10⁴ cells. We further validated this method by detecting a DHS site of the IL-4 gene that is specifically present in type 2 but not type 1 T helper cells. Overall, this method overcomes the interference by genomic DNA not cut by DNase I at the DHS site, thereby offering unambiguous detection of DHS sites in the cells.     

Delineation of AbrB-binding sites on the Bacillus subtilis spo0H, kinB, ftsAZ, and pbpE promoters and use of a derived homology to identify a previously unsuspected binding site in the bsuB1 methylase promote.

DNase I footprinting experiments showed that AbrB binds to the regulatory regions of the spo0H, kinB, ftsAZ, and pbpE genes. A conserved motif was found in these and other AbrB-binding sites. A search for Bacillus subtilis DNA sequences containing this motif led to the prediction that AbrB would bind to the promoter controlling the bsuB1 methylase gene. DNase I footprinting experiments confirmed this prediction.