The change of DNA structure by specific binding of the cAMP receptor protein from rotation diffusion and dichroism measurements.

The structure of complexes formed between cAMP receptor protein (CRP) and various restriction fragments from the promoter region of the lactose operon has been analysed by measurements of electrodichroism. Binding of CRP to a 62-bp fragment containing the major site leads to an increase of the rotation time constant from 0.33 to 0.43 microseconds; addition of cAMP to the complex induces a decrease to 0.25 microseconds. Similar data are obtained for a 80-bp fragment containing the operator site; however, in this case the decrease of the rotation time for the specific complex is only observed when the salt concentration is increased from 3 to 13 mM. A 203-bp fragment containing both sites showed a corresponding change after pre-incubation at 50 mM salt. The salt dependence of the rotation time for the specific complex formed with the 203-bp fragment also indicates that a compact structure is formed at 13 mM salt, whereas the structure is not as compact at 3 mM salt. A 98-bp fragment without specific CRP sites did not reveal changes corresponding to those observed for the specific fragments. The rotation time constants together with the dichroism amplitudes indicate that binding of CRP to specific sites in the presence of cAMP leads to the formation of compact structures, which are consistent with bending of DNA helices. The observed strong salt dependence of the structure is apparently due to electrostatic repulsion between adjoining helix segments.     

Equilibrium studies of the cyclic AMP receptor protein-DNA interaction

The binding of the Escherichia coli cyclic AMP receptor protein (CAP) to restriction fragments containing the lac promoter-operator region has been investigated as a function of cAMP concentration, using a sensitive gel electrophoresis assay. Under standard conditions (13 mM ionic strength), the equilibrium constant for CAP binding to its primary site on a 203 base-pair lac promoter fragment is 6.3 X 10(8) M-1 at 0.2 microM-cAMP, and increases to 8.4 X 10(10) M-1 at 5.0 microM-cAMP. The latter is about 10(5) times larger than the equilibrium constant for binding to an isolated, non-specific site. The L8 mutation, which renders the lac promoter unresponsive to CAP in vivo, lowers this binding affinity by five- to tenfold. Analysis of the cAMP dependency of binding over the concentration range of 0.2 microM to 10 microM reveals that uptake of a single equivalent of cAMP is required for site-specific binding. Similarly, the transfer of CAP from a non-specific DNA site to a specific site requires the net uptake of a single molecule of cAMP. In contrast, co-operative non-specific binding to DNA was found to be independent of cAMP concentration with an equilibrium binding constant of 6 X 10(6) M-1. We conclude that the cAMP affinity of the two CAP subunits in the specific promoter complex is not equal, and that the complex structure therefore deviates significantly from twofold symmetry. A model for the regulation of the lac promoter by the intracellular cAMP concentration is proposed on the basis of the equilibrium binding results.     

On the origin of selectivity in recognition by cyclic adenosine 3',5'-monophosphate receptor protein of its specific binding site of the lactose promoter region

From fluorescence measurements we could analyse the binding of cyclic adenosine 3',5'-monophosphate receptor protein (CRP) from Escherichia coli to its specific site on a 301 base-pair long DNA fragment containing the control region of the lactose operon. At physiological ionic strength selection of the specific site is strictly dependent on the allosteric effector cAMP, and binding of the cAMP . CRP complex to its specific site is favoured over the non-specific binding by 5 kcal/mol with Kass (specific) = 10(8) M-1 at 37 degrees C.     

Ligand-modulated binding of a gene regulatory protein to DNA. Quantitative analysis of cyclic-AMP induced binding of CRP from Escherichia coli to non-specific and specific DNA targets

This paper describes a generally applicable method for quantitative investigation of ligand-dependent binding of a regulatory protein to its target DNA at equilibrium. It is used here to analyse the coupled binding equilibria of cAMP receptor protein from Escherichia coli K12 (CRP) with DNA and the physiological effector cAMP. In principle, the DNA binding parameters of CRP dimers with either one or two ligands bound are determinable in such an approach. The change of protein fluorescence was used to measure CRP binding to its recognition sequence in the lac control region and to non-specific DNA. Furthermore, the binding of cAMP to preformed CRP-DNA complexes was independently studied by equilibrium dialysis. The data were analysed using a simple interactive model for two intrinsically identical sites and site-site interactions. The intrinsic binding constant K and the co-operativity factor alpha for binding of cAMP to free CRP depend only slightly on salt concentration between 0.01 M and 0.2 M. In contrast, the affinity of cAMP for CRP pre-bound to non-specific DNA increases with the salt concentration and the co-operativity changes from positive to negative. This results from cation rebinding to the DNA lattice upon forming the cAMP-CRP-DNA complex from cAMP and the pre-formed CRP-DNA complex. The CRP-cAMP1 complex shows almost the same affinity for specific and non-specific DNA as the CRP-cAMP2 complex, and both displace the same number of cations. It is concluded that the allosteric activation of CRP is induced upon binding of the first cAMP. These results are used to estimate the occupation of the CRP site in the lac control region in relation to the cAMP concentration in vivo. Under physiological conditions the lac promoter is activated by the CRP dimer complexed with only one cAMP. Furthermore, a model for the differential activation of various genes expressed under catabolite repression is presented and discussed.     

Sensitization of the Escherichia coli cyclic AMP receptor protein to trypsin cleavage by polydeoxyribonucleotides and polyribonucleotides

In the absence of cAMP the cyclic AMP receptor protein (CRP) is relatively resistant to trypsin whereas the cAMP X CRP complex is attacked yielding N-terminal core fragments of 14,300 and 18,500 Da which still bind cAMP. The DNA X CRP complex formed at low ionic strength in the absence of cAMP is cleaved by trypsin with the formation of 9,700- and 6,000-Da fragments and the concomitant loss of cAMP binding activity. DNA X CRP remains as resistant to attack by subtilisin, clostripain, and the Staphylococcus aureus V8 protease as unliganded CRP but is slowly digested by chymotrypsin. All of the double-stranded polydeoxyribonucleotides and several of the single-stranded polydeoxyribonucleotides and polyribonucleotides tested render CRP sensitive to cleavage by trypsin. CRP is less rapidly cleaved by trypsin in the presence of d(A)n, d(I)n, and r(C)n indicative of a weaker affinity of CRP for these polynucleotides. The 9,700-Da fragment is N-terminal in CRP and probably terminates at Lys-89. The loss of cAMP binding activity following trypsin cleavage of DNA X CRP indicates that regions beyond this residue are important in the function of the cAMP-binding domain of CRP. The 6,000-Da fragment extends from Val-131 to Arg-185 or Lys-188 and contains part of the F helix involved in DNA binding by CRP.     

Allosteric control of cAMP receptor binding dynamics

The intrinsic fluorescence of the cyclic AMP receptor is a sensitive indicator of the reaction with DNA, but signals are perturbed by a photoreaction. A ratio procedure is shown to be useful for correction. The reaction of the protein with DNA indicated by corrected transients extends over a broad time range not only at low salt concentrations but also at physiological salt concentrations. The initial binding step can be recorded preferentially at low salt pH 7 and is shown to be very similar for specific and nonspecific DNA. The rate constant for initial binding at 13.5 mM salt pH 7 is 2 × 10(8) M(-1) s(-1). Slow reaction steps up to times of several hundred seconds are observed both at low and high salt; the magnitude and sign of fluorescence amplitudes are strongly dependent on salt and pH. At 100 mM salt pH 8, the slow reaction step observed for the binding of the cyclic AMP receptor protein to promoter DNA is strongly shifted to longer times upon reduction of the cAMP concentration. The observed cAMP dependence is described quantitatively by a model implying that binding of the receptor to promoter DNA requires two cAMP molecules per protein dimer and is not consistent with a model assuming that a single cAMP is sufficient for activation. The rate constant for binding of the protein·dimer·(cAMP)(2) complex to the promoter is 1.3 × 10(8) M(-1) s(-1), close to the limit of diffusion control. Equilibration of specific complexes takes ~100 s at physiological concentrations of the reaction components.     

Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities

Cyclic AMP receptor protein (CRP) from Escherichia coli is assumed to exist in two states, namely, those represented by the free protein and that of the ligand-protein complex. To establish a quantitative structure-function relation between cAMP binding and the cAMP-induced conformational changes in the receptor, protein conformational change was quantitated as a function of cAMP concentration up to 10 mM. The protein conformation was monitored by four different methods at pH 7.8 and 23 degrees C, namely, rate of proteolytic digestion by subtilisin, rate of chemical modification of Cys-178, tryptophan fluorescence, and fluorescence of the extrinsic fluorescence probe 8-anilino-1-naphthalenesulfonic acid (ANS). Each of these techniques reveals a biphasic dependence of protein conformation on cAMP concentration. At low cAMP concentrations ranging from 0 to 200 microM, the rates of proteolytic digestion and that of Cys-178 modification increase, whereas the fluorescence intensity of the ANS-protein complex is quenched, and there is no change in the fluorescence intensity of the tryptophan residues in the protein. At higher cAMP concentrations, the rates of proteolytic and chemical modification of the protein decrease, while the fluorescence intensity of the ANS-protein complex is further quenched but there is an increase in the intensity of tryptophan fluorescence. These results show unequivocally that there are at least three conformational states of the protein. The association constants for the formation of CRP-cAMP and CRP-(cAMP)2 complexes derived from conformational studies are in good agreement with those determined by equilibrium dialysis, nonequilibrium dialysis, and ultrafiltration. Therefore, the simplest explanation would be that the protein exhibits three conformational states, free CRP and two cAMP-dependent states, which correspond to the CRP-cAMP and CRP-(cAMP)2 complexes. The binding properties of CRP-cAMP and CRP-(cAMP)2 to the lac promoter were studied by using the gel retardation technique. At a high concentration of cAMP which favors the formation of the CRP-(cAMP)2 complex, binding of the protein to DNA is decreased. This, together with conformational data, strongly suggests that only the CRP-cAMP complex is active in specific DNA binding whereas CRP and CRP-(cAMP)2 are not.     

Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli

Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes in Escherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long-distance signal transmission from the N-terminal cAMP-binding domain to the C-terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this report we have used a CRP mutant containing a single Trp13 located in the N-terminal domain of the protein. We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule. The results presented provide evidence for the occurrence of a long-distance conformational signal transduction within the protein from the C-terminal DNA-binding domain to the N-terminal domain of CRP. This conformational signal transmission depends on the promoter sequence. We also used the stopped-flow and Forster resonance energy transfer between labeled Cys178 of CRP and fluorescently labeled DNA sequences to study the kinetics of DNA-CRP interactions. The results thus obtained lead to the conclusion that CRP can exist in several conformational states and that their distribution is affected by binding of both the cAMP and of specific DNA sequences.     

Specific DNA binding of the cyclic AMP receptor protein to a synthetic oligodeoxyribonucleotide. A circular dichroism study

The interaction of the cAMP receptor protein (CRP) of Escherichia coli with a synthetic DNA undecamer (11 mer) comprising a portion of the specific target site in the gal operon and containing 8 basepairs out of the 10 basepair concensus making up specific CRP sites, has been studied by circular dichroism spectroscopy. The binding constants for the interaction of CRP with the 11 mer in the presence and absence of cAMP have been determined, and it is shown that CRP, both in the presence and absence of cAMP, induces a B-C transition in the conformation of the 11 mer.     

Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay

DNA binding properties of the proteins required for induction of the Escherichia coli L-arabinose operon were measured using a polyacrylamide gel electrophoresis assay. The mechanisms of induction and repression were studied by observing the multiple interactions of RNA polymerase, cyclic AMP receptor protein and araC protein with short DNA fragments containing either the araC or araBAD promoter regions. These studies show that binding of araC protein to the operator site, araO1, directly blocks RNA polymerase binding at the araC promoter, pC. We find that cyclic AMP receptor protein and araC protein do not bind co-operatively at their respective sites to linear DNA fragments containing the pBAD promoter. Nevertheless, both these positive effectors must be present on the DNA to stimulate binding of RNA polymerase. Additionally, binding of the proteins to the DNA is not sufficient; araC protein must also be in the inducing state, for RNA polymerase to bind. Equilibrium binding constraints and kinetics were determined for araC protein binding to the araI and the araO1 sites. In the presence of inducer, L-arabinose, araC protein binds with equal affinity to DNA fragments containing either of these sites. In the presence of anti-inducer, D-fucose, the affinity for both sites is reduced 40-fold. The apparent equilibrium binding constants for both states of the protein vary in parallel with the buffer salt concentration. This result suggests that the inducing and repressing forms of araC protein displace a similar number of cations upon binding DNA.     

Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector

The binding of a protein to its specific sequence, borne on a DNA fragment, retards the mobility of the fragment in a characteristic way during gel electrophoresis. If the protein induces bending in the DNA, the contortion can also be monitored by gel electrophoresis, because the amount of retardation of the mobility of the DNA-protein complex is dependent upon the position and the degree of the bend induced in the DNA fragment [Wu and Crothers, Nature 308 (1984) 509-513]. We have constructed a plasmid, pBend2, which can generate a large number of DNA fragments of identical length in which the protein-binding nucleotide sequence is located in circular permutations. The vector contains two identical DNA segments containing 17 restriction sites in a direct repeat spanning a central region containing cloning sites. The protein-binding sequence is inserted at one of these cloning sites. To investigate the functional significance of bending, we have compared, using pBend2, the cAMP.cAMP-receptor protein (CPR)-induced bending of CRP-binding sites found in five different genes of Escherichia coli. We have also shown that the bacteriophage lambda 0R1 operator DNA is bent when complexed with the CI or Cro repressor of the phage.