Influence of DNA structure on the lactose operator-repressor interaction (original) (raw)
Related papers
Biochemistry, 1977
The stoichiometries of binding of non-operator DNA and inducer to lac repressor, as well as some conformational aspects of these interactions, are described in this paper. It is shown that the circular dichroism (CD) spectrum of the repressor-non-operator DNA complex is appreciably different from that obtained by summing the spectra of the separate components; the major change is a substantial enhancement of the positive (-275 nm) lobe of the originally conservative DNA B form pattern. These CD spectral changes appear to reflect a change in DNA conformation on repressor binding and are interpreted in terms of tilting (relative to the DNA axis) of some of the base pairs of the native structure, or perhaps some twisting of the overall structure resulting in a tighter coupling of vicinal base transition moments. These changes in the CD spectrum of non-operator DNA on repressor binding have been monitored as a function of added repressor concentration, under tight-binding conditions, to establish that the site size (n) for binding to non-operator DNA is -12 base pairs per repressor tetramer (-24 base pairs if repressor binds to both sides of the double helical DNA lattice). This value of n is confirmed both by calculation from binding isotherms , Biochemistry 16 (following paper in this issue), and by titration of repressor T h e lac repressor recognizes (and binds tightly to) a particular sequence of base pairs in the Escherichia coli chromosome which is defined, on this basis, as the lac operator. By virtue of this specific binding the repressor exerts negative control over the expression of the lactose operon (Jacob and . The interaction of lac repressor with operator has been subjected to intense scrutiny by both biochemical and genetic means, and a great deal of information is now available State University, East Lansing, Mich. 48824. sulfhydryl groups as a function of added DNA concentration. The measured site size is discussed in terms of the relationship between operator and non-operator DNA binding of repressor, and of various features of the known operator sequence, to suggest alternative models for the geometry of the repressoroperator interaction. Investigation of the repressor-inducer interaction by equilibrium dialysis, fluorescence, and gel permeation chromatography shows, in confirmation of the results of Ohshima, Y., et al. , that different repressor preparations exhibit different (average) numbers of "active" inducer binding sites per repressor tetramer (nl); values of nl between two and four have been obtained. The effects on n~ of a variety of environmental conditions have been examined, and the results, together with relevant data from the literature, are discussed in terms of conformational equilibria between forms of repressor subunits which bind strongly to inducer and weakly to operator (RI), and forms which bind strongly to operator and weakly to inducer (Ro). Thermodynamic parameters for the binding of inducer to repressor subunits (in the RI form) have also been determined. At pH 7.6 (4-25 "C), AGO = -7.7 kcal/mol (25 "C), AH" = -6.2 kcal/mol, and AS" = +5 cal mol-' deg-' for this reaction. about both the protein and the operator (for a recent review, see .
Biochemistry, 2008
Many mutations that impact protein function occur at residues that do not directly contact ligand. To understand the functional contributions from the sequence that links the DNA-binding and regulatory domains of the LacI/GalR homologues, we have created a chimeric protein (LLhP), which comprises the LacI DNA-binding domain, the LacI linker, and the PurR regulatory domain. Although DNA binding site residues are identical in LLhP and LacI, thermodynamic measurements of DNA binding affinity show that LLhP does not discriminate between alternative DNA ligands as well as LacI. In addition, small-angle scattering experiments show that LLhP is more compact than LacI: Upon DNA release, LacI shows a 20Å increase in length that was previously attributed to unfolding the linker. This change is not seen in apo-LLhP, even though the linker sequences of the two proteins are identical. Together, results indicate that long-range functional and structural changes are propagated across the interface that forms between the linker and regulatory domain. These changes could be mediated via the side chains of several linker residues that contact the regulatory domains of the naturally-occurring proteins, LacI and PurR. Substitution of these residues in LLhP leads to a range of functional effects. Four variants show altered affinity for DNA, with no changes in selectivity or allosteric response. Another two result in proteins that bind operator DNA with very low affinity and no allosteric response, similar to LacI binding nonspecific DNA sequences. Two more substitutions simultaneously diminish affinity, enhance allostery, and profoundly alter DNA ligand selectivity. Thus, positions within the linker can be varied to modulate different aspects of repressor function.
DNAase footprinting a simple method for the detection of protein-DNA binding specificity
Nucleic Acids Research, 1978
A method for studying the sequence-specific binding of proteins to, DNA is described. The technique is a simple conjoining of the Maxam-Gilbert DNA-sequencing method and the technique of DNAase-protected fragment isolation. Fragments of a 5' end-labelled, double-stranded DNA segment, partially degraded by DNAase in the presence and absence of the binding protein, are visualized by eletrophoresis and autoradiography alongside the base-specific reaction products of the Maxam-Gilbert sequencing method. It is then possible to see the protective "footprint" of the binding protein on the DNA sequence. The binding of lac repressor to lac operator is visualized by "footprinting" as an example. Equilibrium estimates indicate that 10-fold sequence-specificity (differential binding constant) could be studied easily using this technique. tages and limitations. CX Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
Nucleic Acids Research, 1989
An electrophoretic procedure for the measurement of the helix unwinding induced by a sequence-specific protein is described. The method, which was applied here to EcoR I, CAP and lac repressor, involved the migration of the complexes with positively and negatively supercoiled DNA minicircles carrying a single protein binding site. Mobility shifts of complexes relative to naked DNAs appeared to be a result of i) the unwinding; of ii) an increase in the molecular frictional coefficient, which led to a retardation; of iii) bending, in the particular case of CAP, which induced an acceleration; and of iv) looping, in the case of lac repressor, which also resulted in an acceleration. Under conditions where the migration of the naked topoisomers was V-like (topoisomer mobility showed the same linear increase with both negative and positive supercoilings; Zivanovic et al. (1986) J. Mol. Biol., 192, 645-660), the protein unwinding contribution to mobility was assumed to be identical to that experimentally observed in the case of a thermal unwinding: all negatively supercoiled topoisomers were retarded and all positively supercoiled topoisomers were accelerated to the same extent. In contrast, the mobility contribution of the frictional term, as well as those of bending and looping, appeared to vary strongly with the magnitude of the supercoiling, but only weakly with its polarity. As a consequence, these latter contributions may approximately cancel when one is measuring the difference between the shifts observed for two comigrating, negatively and positively supercoiled, topoisomers, allowing the unwinding to be calculated. While estimates obtained for EcoR I, 23 ± 3°, and CAP, about 29°, were in good agreement with previous measurements using topoisomerase 1, the value found for lac repressor, 13 to 160, was significantly smaller. with a plasmid DNA molecule usually containing several protein binding sites in order to increase the accuracy. This relaxation (or closure), when compared to that of the naked plasmid, allowed the determination of U as the increment in the linking number of the most probable configuration of the DNA. This method has initially been applied to E. coli RNA polymerase by Saucier & Wang (1), and was subsequently used with lac repressor (2), restriction endonuclease Eco R (3), catabolite gene activator protein (CAP) (4), Xenopus transcription factor IIIA (5), and again E. cofi RNA polymerase (6). Another method, which has been of more limited use, takes advantage of the property of a protein with an ability to unwind the DNA to exhibit a higher affinity for DNA of larger negative supercoiling. Quantification of this effect, together with the knowledge of the free energy of superhelix formation, has permitted an estimation of the unwinding angle of lac repressor (7) and of E. cofi RNA polymerase (6).
DNA Conformation and Transcription
2005
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The interaction of RNA polymerase and lac repressor with the lac control region
Nucleic Acids Research, 1979
We have examined the interactions of lac repressor and RNA polymerase with the DNA of the lac control region, using a method for direct visualization of the regions of DNA protected by proteins from DNAase attack. The repressor protects the operator essentially as reported by Gilbert and Maxam (1) with some small modifications. However, the evidence reported here concerning the binding of RNA polymerase to the DNA of the promoter mutant UV5 indicates that : 1) the RNA polymerase molecule binds asymmetrically to the promoter DNA, 2) RNA polymerase protects DNA sequences to within a few bases of the CAP binding site, suggesting direct interaction between polymerase and the CAP protein at this site, 3) RNA polymerase still binds to the promoter when repressor is bound to the operator, but fails to form the same extensive complex.
Nucleic Acids Research, 1976
The binding of lactose repressor to non-operator DNA was studied by the modification of several DNA's, including glycosylated DNA, with dimethyl sulphate, which affects the minor and major grooves of DNA and single stranded DNA regions. The non-specific binding of the repressor to DNA protected the minor groove but apparently not the major groove of the DNA double helix against methylation and did not increase the content of single stranded DNA regions. This suggests that the repressor on binding to non-operator DNA makes contacts mainly in the minor groove of DNA and does not uncoil the DNA double helix. This is different from the interaction of the represaor with lactose operator DNA which occurs, as shown by Gilbert et_al. (l), along both the major and the minor groove. I. ( K = 10" 5 M) (5-6). Here we report the results of methylation with dimethyl sulphate of the complex of the lac repressor with non-operator DNA. The results show that the repressor protects the minor but apparently not the major groove O-lnformation Retrieval Limited 1 Falconberg Court London W1V 5FG England by guest on August 18, 2016 http://nar.oxfordjournals.org/ Downloaded from
Binding of synthetic lactose operator DNAs to lactose repressors
Proceedings of the National Academy of Sciences, 1977
The nitrocellulose filter assay was used to study the interactions of wild-type (SQ) and tight-binding (QX86) lac repressors with synthetic lac operators 21 and 26 base pairs long. The repressor binding properties of both operators were very similar, indicating that both contain the same specific repressor recognition sites. The repressor-operator association rate constants (ka) were more sensitive than dissociation rate constants (kd) to changes in ionic strength. The responses of both ka and kd to ionic strength were relatively small compared to the effects previously observed with XhS0dlac as operator DNA. These results suggest that under natural conditions there are electrostatic interactions between lac repressor and DNA regions outside of the 26 base pair operator sequence. Association rate constants for SQ repressor with either operator are higher than have been predicted for diffusion-limited reactions. We postulate that long-range electrostatic attractions between repressor and operator accelerate the association reaction. The presence Proc. Natl. Acad. Sci. USA 74 (1977) Vi ew publ i cati on stats Vi ew publ i cati on stats
Tightly bound to DNA proteins: Possible universal substrates for intranuclear processes
Gene, 2012
comb proteins to DNA (Fahrner Q5 and Baylin, 2003). Transition of 66 the chromatin to an active form is preceded by release of histone 67 H1 from internucleosomal DNA and its replacement by high 68 mobility group proteins (HMG; Catez et al., 2004). Besides the 69 rather labile interaction of DNA with histones and HMG proteins, 70 much more stable complexes are also important for regulation of 71 gene activity. Tight bonds, resistant to treatment with salts and Fig. 1. Methods of TBP purification. Afractionation on nitrocellulose; B -DNAse treatment. detergents enable anchorage of chromatin loops to the nuclear 73 matrix. These complexes are important for structuring of the cell 74 nucleus, moreover they are involved in DNA replication, tran-75 scription, repair and recombination ( Q6 Kantidze and Razin, 2009; 76 Podgornaya et al., 2003; Razin et al., 2004, 2007; Stein et al., 77 2003; Taddei et al., 2004). Besides complexes of DNA with nuclear 78 matrix proteins even stronger, sometimes covalent, interactions 79 are formed with another group of proteins usually called tightly 80 bound proteins (TBPs). Operationally this protein fraction is de-81 fined as proteins remaining attached to DNA after its purification 82 by conventional deproteinization methods (lysis in SDS with or