Tet repressor binding induced curvature of tet operator DNA.

Tet repressor dimer binds to two tet operator sites spaced by 30 bp in the Tn10 encoded tet regulatory DNA. The effect of repressor binding on the gel mobility of circular permutated DNA fragments containing either one or both operator sequences is reported. The EcoRI induced bending of DNA is used to compare the results with other protein binding induced structural perturbations of DNA. Tet repressor bends a DNA fragment with a single tet operator to an angle of 42 degrees +/- 7 degrees. The apparent bend angle of DNA fragments containing the tandem tet operator arrangement occupied by two Tet repressor dimers turns out to be 52 degrees +/- 9 degrees. These results are interpreted with respect to the end to end distances of the bent DNA fragments. They indicate that either the intervening tet regulatory DNA between the operators or the bound operator sequences themselves contain additional perturbations from the canonical B-DNA structure. This finding is discussed in the light of previously obtained results from CD, neutron scattering, and electrooptical studies.     

Bending of synthetic bacteriophage 434 operators by bacteriophage 434 proteins.

The extent of DNA bending induced by 434 repressor, its amino terminal DNA binding domain (R1-69), and 434 Cro was studied by gel shift assay. The results show that 434 repressor and R1-69 bend DNA to the same extent. 434 Cro-induced DNA bends are similar to those seen with the 434 repressor proteins. On approximately 265 base pair fragments, the cyclic AMP receptor protein of Escherichia coli (CRP) produces larger mobility shifts than does 434 repressor. This indicates that the 434 proteins bend DNA to a much smaller extent than does CRP. The effects of central operator sequence on intrinsic and 434 protein-induced DNA bending was also examined by gel shift assay. Two 434 operators having different central sequences and affinities for 434 proteins display no static bending. The amount of gel shift induced by 434 repressor on these operators is identical, showing that the 434 repressor bends operators with different central sequences to the same extent. Hence, mutations in the central region of the operator do not influence the bent structure of the unbound or bound operator.     

Interaction of the trp repressor with trp operator DNA fragments

The interaction of the trp repressor with several trp operator DNA fragments has been examined by DNA gel retardation assays and by circular dichroism, in the absence and presence of the corepressor l-tryptophan. The holorepressor binds stoichiometrically to both the trpO and aroH operators, forming 1:1 complexes. In the presence of excess protein, additional complexes are formed with these operator fragments. The relative electrophoretic mobilities of the 1:1 complexes differ significantly for trp and aroH operators, indicating that they differ substantially in gross structure. A mutant trp operator, trpO ^c, has low affinity for the holorepressor, and forms only complexes with stoichiometries of 2:1 (repressor: DNA) or higher, which have a very low electrophoretic mobility. Specific binding is also accompanied by a large increase in the intensity of the near ultraviolet circular dichroism, with only a small blue shift, which is consistent with significant changes in the conformation of the DNA. Large changes in the chemical shifts of three resonances in the ³¹P NMR spectrum of both the trp operator and the aroH operator occur on adding repressor only in the presence of L-tryptophan, consistent with localised changes in the backbone conformation of the DNA.Abbreviations CD circular dichroism - trpO, trpR aroH trp operator fragments - trpO ^c trpMH mutant trp operator fragments     

Protein-induced bending of the simian virus 40 origin of replication

A 3.5 S protein, isolated from mammalian nuclei, specifically binds to DNA fragments containing the simian virus 40 (SV40) origin of replication. Two distinct nucleoprotein complexes are formed, a complex with high electrophoretic mobility carrying probably only one protein molecule, and a complex with reduced electrophoretic mobility carrying probably two protein molecules per DNA fragment. Band shift competition as well as methylation interference assays locate the binding site of the protein in the A + T-rich "late" region of the origin between SV40 nucleotides 13 and 35. The late origin binding (LOB) protein and T antigen bind simultaneously to adjacent sites in the origin. Using circularly permuted DNA fragments of identical lengths we show that the LOB protein induces pronounced bending of the origin fragment. The bending center maps at the 5' end of the adenine tract with one bound protein molecule and at the 3' end when two LOB proteins are bound to one origin fragment.     

Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences.

The Escherichia coli Trp repressor binds to promoters of very different sequence and intrinsic activity. Its mode of binding to trp operator DNA has been studied extensively yet remains highly controversial. In order to examine the selectivity of the protein for DNA, we have used electromobility shift assays (EMSAs) to study its binding to synthetic DNA containing the core sequences of each of its five operators and of operator variants. Our results for DNA containing sequences of two of the operators, trpEDCBA and aroH are similar to those of previous studies. Up to three bands of lower mobility than the free DNA are obtained which are assigned to complexes of stoichiometry 1 : 1, 2 : 1 and 3 : 1 Trp repressor dimer to DNA. The mtr and aroL operators have not been studied previously in vitro. For DNA containing these sequences, we observe predominantly one retarded band in EMSA with mobility corresponding to 2 : 1 complexes. We have also obtained retardation of DNA containing the trpR operator sequence, which has only been previously obtained with super-repressor Trp mutants. This gives bands with mobilities corresponding to 1 : 1 and 2 : 1 complexes. In contrast, DNA containing containing a symmetrized trpR operator sequence, trpRs, gives a single retarded band with mobility corresponding solely to a 1 : 1 protein dimer-DNA complex. Using trpR operator variants, we show that a change in a single base pair in the core 20 base pairs can alter the number of retarded DNA bands in EMSA and the length of the DNase I footprint observed. This shows that the binding of the second dimer is sequence selective. We propose that the broad selectivity of Trp repressor coupled to tandem 2 : 1 binding, which we have observed with all five operator sequences, enables the Trp repressor to bind to a limited number of sites with diverse sequences. This allows it to co-ordinately control promoters of different intrinsic strength. This mechanism may be of importance in a number of promoters that bind multiple effector molecules.     

Mode of DNA binding by SopA protein of Escherichia coli F plasmid

The binding of SopA to the promoter region of its own gene, in which four copies of SopA's recognition sequence, 5'-CTTTGC-3', are arrayed asymmetrically, was examined in vitro. Titration using electrophoretic mobility shift assay showed that the stoichiometry of SopA protomers to the promoter-region DNA is 4 and that the binding is highly co-operative. The co-operativity was corroborated by EMSA and DNase I footprinting for a number of mutant DNA fragments in which 5'-CTTTGC-3' was changed to 5'-CTTACG-3'. EMSA in the style of circular permutation showed that SopA bends DNA. Mutation at either outermost binding site had a different effect on DNA bending by SopA, reflecting the asymmetry in the arrangement of the binding sites, for which the results of DNase I footprinting were in agreement. Gel filtration chromatography and analytical ultracentrifugation of free SopA showed that the protein can exist as a monomer and oligomers in the absence of ATP. Hence, the results indicate that the co-operativity in SopA's DNA binding is based on its intrinsic protein-protein interaction modified by DNA interaction.     

DNA bending near the replication origin of IncFII plasmid NR1.

The DNA replication origin of plasmid NR1 is located approximately 190 base pairs downstream from the 3' end of the repA1 gene, which encodes the essential initiation protein for replication of the plasmid. Restriction endonuclease fragments that contain the NR1 replication origin and its flanking sequences at circularly permuted positions were obtained by digesting oligomers of ori-containing DNA fragments with sets of enzymes that each cut only once in every ori fragment. Polyacrylamide gel electrophoresis of these permuted restriction fragments showed anomalous mobilities, indicating the presence of a DNA bending locus. Through analysis of the relative mobility plots of these permuted fragments, we found one or two possible DNA bending sites located in the intervening region between the repA1 gene and the replication origin of NR1. It seems possible that DNA bending in this region might help to orient the replication origin alongside the repA1 gene, which could contribute to the cis-acting character of the RepA1 initiation protein.     

Angle and locus of the bend induced by the msp I DNA methyltransferase in a sequence-specific complex with DNA.

Bending of DNA induced by M.Msp I, one of the m5C-DNA methyltransferases, has been investigated using circular permutation analysis. The M.Msp I MTase induced sharp bends in DNA containing its recognition sequence 5'-CCGG-3'which was estimated to be 142+/-4 degrees and 132+/-4 degrees for circularly permuted DNA fragments of 127 and 1459 bp respectively. The bend centre was found to be asymmetric with respect to the CCGG sequence and appeared to exclude the 'target cytosine'. An estimate of approximately 15 kcal/mol was obtained for the free energy associated with M.Msp I-induced DNA bending.     

LexA repressor induces operator-dependent DNA bending

LexA, the repressor of the SOS system in Escherichia coli induces a substantial DNA bending upon interaction with the operator of the caa gene, which codes for the bacterial toxin colicin A. Analysis by gel electrophoresis of a family of DNA fragments of identical length, but bearing the caa operator at different positions, shows that DNA bending occurs close to or within the operator sequence upon LexA binding. In contrast, the interaction of LexA with the recA operator induces no detectable bending on 5% polyacrylamide gels. This difference between the two operators is likely to be due to an intrinsic bendability of the caa operator related to thymine tracts located on both sides of the operator. Such tracts do not exist in the recA operator. The free DNA fragments harbouring the caa operator show a slight tendency to bend even in the absence of the LexA repressor. The centre of this intrinsic bend is located close to or within the caa operator.     

The sequence-directed bent DNA detected in the replication origin of Chlamydomonas reinhardtii chloroplast DNA is important for the replication function

Summary We demonstrated that the 1055 by restriction fragment containing OriA, a chloroplast DNA replication origin of Chlamydomonas reinhardtii, has electrophoretic anomalies characteristic of bent DNA. A tandem dimer of the region was constructed. Quantitative measurement of the relative gel mobility of a set of permuted fragments was used to extrapolate the approximate position of the bent DNA segment. By analyzing the gel mobility of short, sequenced fragments of the bent DNA region, the putative bending locus was identified. Two A₄ tracts and two A5 tracts were located in the bending locus. Oligonucleotide-directed mutagenesis was then used to disrupt the A tract or the spacing between A tracts and the effect of site-specific mutation on electrophoretic mobility was analyzed. To assess the functional role of the bent DNA region, subclones containing the bending locus, mutated bending locus, and regions flanking the bending locus were constructed. Each subclone was used as template in an in vitro DNA replication system which preferentially initiated DNA replication at OriA. A 224 by subclone with the bending locus positioned in the middle displayed the highest replication function and was sufficient to initiate DNA replication in vitro. Site-specific mutations or alterations of the A tracts resulted in decreased DNA bending and decreased DNA replication activity.     

A control element within a structural gene: the gal operon of Escherichia coli

The gal operon of Escherichia coli is transcribed from two overlapping promoters, PG1 and PG2. Cyclic AMP and its receptor protein (CRP) modulate the two promoters in opposite directions by binding to a single cat locus. Both the promoters are negatively regulated by a single repressor, the product of the galR gene. An operator site, defined by several mutations, has previously been located upstream from the cat locus. We have isolated and characterized a new set of cis-dominant constitutive mutations of the gal operon and determined their locations by DNA sequencing. From these studies, we propose the existence of a second functional gal operator element at an extraordinary site--within galE, the first structural gene. Both the operators, OE (exterior) and OI (interior), are involved in the repression of PG1 and PG2. This would be the first example of the presence of a functional operator element within a structural protein-coding region.     

lac repressor forms stable loops in vitro with supercoiled wild-type lac DNA containing all three natural lac operators

We have analyzed protein-DNA complexes formed between lac repressor and linear or differently supercoiled lac DNA (802 or 816 base-pairs in length), which carry all three natural lac operators (O1, O2 and O3) in their wild-type sequence context and spacing and compared them with constructs that contain specifically mutated "pseudo-operators" O2 or O3. We used gel retardation assays to identify the nature of the complexes according to their characteristic electrophoretic mobility and dissociation rate measurements to determine their stability. With linear DNA we found only indirect evidence for loop formation between O1 and O2. In covalently closed DNA minicircles the formation of a loop between O1 and O2 could be demonstrated by the observation that O1-O2 containing DNA with low negative supercoiling (sigma = -0.013 and less) is constricted by binding of lac repressor, resulting in an increased electrophoretic mobility. At elevated negative supercoiling (sigma = -0.025, -0.037, -0.05) O1-O2 containing DNA complexed with lac repressor migrates significantly slower than the corresponding O1-DNA, indicating loop formation. The dissociation of lac repressor-operator complexes is decreased with increasing negative supercoiling for all tested operator combinations of O1, O2 and O3. However, in the presence of at least two natural lac operators on the same DNA minicircle the enhancement of stability is particularly large. This indicates that a DNA loop is formed between these two lac operators, O1 and O2 as well as O1 and O3, since negative supercoiling is known specifically to promote the formation of looped structures. Additionally, we observe a dependence of dissociation rate on the spatial alignment of the operators as a result of changing helical periodicity in differently supercoiled DNA and consider this to be further evidence for loop formation between O1 and O2 as well as O1 and O3.