Protein/DNA architecture of the DNase I hypersensitive region of the Drosophila hsp26 promoter.

Genomic footprinting on the Drosophila hsp26 promoter in isolated nuclei has shown that a TATA box binding factor is present before and after induction by heat shock, while three of the seven heat shock consensus sequences 5' of the gene are occupied (presumably by heat shock factor, HSF) specifically on heat shock. The sites of HSF interaction are separated by greater than 200 bp of which approximately 150 bp are bound to the surface of a nucleosome. The juxtaposition of these various macromolecules on the DNA suggests a basis for the major DNase I hypersensitive site 5' of hsp26 and a novel tertiary structure for the promoter complex.     

Interaction of Escherichia coli RNA polymerase holoenzyme containing sigma 32 with heat shock promoters. DNase I footprinting and methylation protection

The DNase I protection pattern of E sigma 32 was assayed on three heat shock promoters, the E sigma 32 promoter for the groESL operon, P2 of the dnaKJ operon, and rpoD PHS, the E sigma 32 promoter upstream from rpoD. E sigma 32 protected each of these promoters from DNase I digestion from around -60 to around +20. Protection from dimethyl sulfate methylation was assayed at the groE promoter. E sigma 32 binding altered the sensitivity to methylation of bases in the vicinity of both the -10 and -35 regions. The DNase I footprints for the E sigma 32 promoters were very similar to the DNase I footprint of E sigma 70 on the lacUV5 promoter. After analyzing the DNase I footprints by taking into account the contacts predicted to be made by DNase I, it appeared that E sigma 32, like E sigma 70, contacts the DNA primarily on one face of the helix in the -35 region and on both faces in the -10 region.     

High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site.

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.     

Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.     

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.     

Footprint analysis of the bsp RI DNA methyltransferase-DNA interaction.

The interaction between the GGCC-specific Bsp RI DNA methyltransferase (M. Bsp RI) and substrate DNA was studied with footprinting techniques using a DNA fragment that was unmodified on both strands. Footprinting with DNase I revealed an approximately 14 bp protected region. Footprinting with dimethylsulfate detected major groove interactions with the guanine bases of the recognition sequence. Reaction with 1,10-phenanthroline-copper did not show protection, suggesting that minor groove interactions play little role in sequence-specific recognition by M. Bsp RI. Hydroxyl radical footprinting revealed a protected stretch of 6 nt. The hydroxyl radical footprint of M. Bsp RI differs markedly from the the footprint reported for the Hha I and Sss I methyltransferases. The pattern of protection from dimethylsulfate and hydroxyl radicals suggests that the interactions of M. Bsp RI with DNA are similar to those detected in the co-crystal structure of the Hae III methyltransferase.     

The beta-globin dominant control region: hypersensitive site 2.

The Dominant Control Region (DCR) of the human beta-globin gene locus consists of four strong hypersensitive sites (HSS) upstream of the epsilon-globin gene. Addition of these sites confers copy number dependent expression on the human beta-globin gene in murine erythroleukaemia cells and transgenic mice, at levels comparable with the endogenous mouse globin genes. We have shown previously that a 1.9 kb fragment comprising HSS 2 accounts for 40-50% of the full effect of the DCR. In this paper we describe a deletional analysis of HSS 2. We show that a 225 bp fragment is sufficient to direct high levels of expression of the human beta-globin gene which is copy number dependent and integration site independent. This 225 bp fragment overlaps the major region that is hypersensitive 'in vivo'. DNase I footprinting shows the presence of four binding sites for the erythroid specific protein NF-E1; the three other footprinted regions display a remarkable redundancy of the sequence GGTGG and bind a number of proteins including Sp1 and the CACC box protein. The significance of these results for the regulation of globin gene expression is discussed.