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Proc. Nat. Acad. Sci. USA Vol. 70, No. 9, pp. 2529-2533, September 1973 Cyclic Adenosine Monophosphate Receptor: Loss of cAMP-Dependent DNA Binding Activity after Proteolysis in the Presence of Cyclic Adenosine Monophosphate (binding assay/DNA-cellulose chromatography/conformation) JOSEPH S. KRAKOW* AND IRA PASTANt * Department of Biological Sciences, Hunter College of CUNY, New York, New York 10021; and t Laboratory of Molecular Biology, National Cancer Intitute, National Institutes of Health, Bethesda, Maryland 20014 Communicated by Severo Ochoa, May 14, 1973 ABSTRACT The cAMP receptor requires cAMP for and was judged to be at least 95% pure by Na dodecyl sul- DNA binding at pH 8.0 but shows cAMP-independent DNA fate-gel electrophoresis. This material was equally as active binding at pH 6.0. Incubation of the cAMP receptor with proteolytic enzymes in the presence ofcAMP results in loss in promoting gal transcription in vitro as cAMP receptor of DNA-binding ability at pH 8, while it is still able to bind prepared by the method of Anderson et al. (1). cAMP and DNA at pH 6. Incubation with proteolytic en- zyme in the absence of cAMP does not affect the DNA-bind- [3H]cAMP Binding. The assay was similar to that of Ander- ing properties of the cAMP receptor. After proteolysis in son et al. (1) with the ammonium sulfate precipitate collected the presence of cAMP, analysis by sodium dodecyl sulfate- on a Whatman GFC glass-fiber filter rather than by centrif- acrylamide-gel electrophoresis shows that the 22,500-dal- ugation. ton subunit characteristic of the untreated protein has been completely replaced by a 12,500-dalton fragment. [3H]d(A-T) or [3H]d(I-C) Binding. To lower the blank ad- of the labeled polymers, the nitrocellulose filters AMP is a sorption The cyclic (cAMP) receptor DNA-binding protein were soaked in 0.1 M KOH for 30 min at room temperature in which the affinity for DNA is dependent on cAMP (1-3). (250) (9) and then placed in 20 mM Tris HCl (pH 7.8)-50 cAMP receptor acts as an auxiliary factor for the RNA poly- filters were where mM NaCl before use. After the binding assay the merase holoenzyme for catabolite-repressible operons in toluene in a Beckman LS 230 requires the coincident binding dried and counted liquifluor initiation of transcription scintillation counter. All incubations were done in polystyrene of both cAMP receptor and RNA polymerase to the promoter in tubes. (4, 5). cAMP receptor is considered an allosteric protein was of in cAMP DNA-cellulose prepared by the method Alberts which cAMP binding elicits a conformational change et al. (10), with heat-denatured calf-thymus DNA. receptor necessary for DNA binding (6). We will present evidence showing that limited proteolytic digestion of cAMP RESULTS receptor in the presence (but not in the absence) of cAMP in Binding of cAMP receptor to DNA containing catabolite results in a modified form of cAMP receptor which the operons requires cAMP (2, 3); this is also true of cAMP receptor repressible characteristic 22,500-dalton subunit native for formation of cAMP receptor-d(A-T) and cAMP receptor- has been replaced by a 12,500 (±500)-dalton fragment. The d(I-C) complexes (Fig. 1). The data show that the cAMP modified cAMP receptor, although able to bind cAMP and concentration required for half-maximum binding varies to form complexes with poly(dA-dT) -poly(dA-dT) [d(A-T) ] with the polydeoxynucleotide assayed. Half-maximum bind- at pH 6.0 (cAMP-independent binding), is unable to carry ing to cAMP receptor occurred at a concentration of 7 X out the cAMP-dependent binding of d(A-T) at pH 8.0 char- 10-7 M cAMP for [PH]d(I-C) and 1 X 10-5 M cAMP for acteristic of native cAMP receptor. [3H]d(A-T). Other experiments have indicated that cAMP MATERIALS AND METHODS receptor + d(I-C) has a higher affinity for cAMP than either cAMP receptor alone or cAMP receptor + d(A-T); this find- Materials. Phenylmethanesulfonylfluoride, dithiothreitol, ing is consonant with the data showing that a low concentra- trypsin, chymotrypsin, subtilisin, cAMP, cGMP, 5'-AMP, tion of cAMP is required for d(I-C) binding. The apparent ADP, ATP, dATP, TTP, dCTP, and Bis-Tris-propane buffer high affinity of cAMP receptor + cAMP for d(I-C) relative were products of Sigma. Nitrocellulose filters (0.45-,um pore to d(A-T) is in contrast to the much higher affinity of the lac size, 25-mm diameter) were obtained from Matheson-Higgins. repressor for d(A-T) relative to d(I-C) (11). These differences Tritiated cAMP, TTP, and dCTP were obtained from New may reflect some properties of the promoter and operator England Nuclear Corp. dITP was prepared by nitrous acid sequence or secondary structure that would be recognized by deamination of dATP by the method of Inman and Baldwin cAMP receptor and lac repressor. (7). Labeled and unlabeled d(A-T) and poly(dI-dC) - poly(dI- The pH response for the formation of d(A-T)- or d(I-C)- dC) [d(I-C)]were prepared with Escherichia coli DNA poly- cAMP receptor complex is interesting (Fig. 2), with the opti- merase (8) with a specific activity of 1000. mum pH for deoxypolymer binding at pH 8.0. As the pH The cAMP receptor used in these studies was prepared from of the binding assay was raised to pH 10, complete loss of E. coli B by a modified procedure to be presented elsewhere, complex formation resulted. RNA polymerase-[3H]d(A-T) Abbreviations: d(A-T), poly(dA-dT).poly(dA-dT); d(I-C), poly- complexes are retained on nitrocellulose filters at pH 10, in- (dI-dC) *poly(d-dC). dicating that the inability of cAMP receptor to bind to de- 2529 Downloaded by guest on October 1, 2021 2530 Biochemistry: Krakow and Pastan Proc. Nat. Acad. Sci. USA 70 (1973) 3.0 3.0 0 0 E E c x, 2.0 dO-C) 2.0 _ a: a: E 1.0 0 I 1.0 d(A-T) 5 10 20 Minutes -7 -0o6 10-5 10-4 cAMP (M) FIG. 3. Time-course for inactivation of cAMP receptor incu- bated with cAMP + subtilisin. The incubations contained (final FIG. 1. Binding of cAMP receptor to d(A-T) or d(I-C) as a volume, 0.25 ml): 40 mM Bis-Tris-propane buffer (pH 8.0), 0.4 function of cAMP concentration. The incubation contained (final mM dithiothreitol, 2 pg of cAMP receptor, and 0.1 pug of subtili- volume, 0.25 ml): 40 mM Bis-Tris-propane buffer (pH 8.0), 2.5 sin; where indicated 10 nmol of cAMP and 4.5 nmol of [3H]- pg of cAMP receptor, 4.5 nmol of [3H]d(A-T) (1944 cpm/nmol) or d(A-T) were added before incubation at 370 for the periods indi- 4.5 nmol of [3H]d(I-C) (1494 cpm/nmol), and cAMP as indicated. cated. In the assay for binding, each tube received 1 nmol of The mixtures were incubated for 5 min at 37°. After addition of phenylmethanesulfonylfluoride, 100 nmol of cAMP, and to the 0.75 ml of 50 mM NaCl, they were filtered onto nitrocellulose sets which lacked it, 4.5 nmol of [3H]d(A-T). The mixtures were membranes. incubated for 5 min at 37°. After addition of 0.75 ml of 50 mM NaCl, they were filtered onto nitrocellulose membranes. oxypolymers is not a property of the filters at alkaline pH. As the pH was lowered to 6.0, there was an increase in the in cAMP receptor required for DNA binding; this conforma- cAMP-independent binding of cAMP receptor to both (dA-T) tion could occur spontaneously at pH 6 in the absence of and d(I-C) to values approaching that obtained for the cAMP cAMP. + cAMP receptor assays at this pH. The data suggest that The data presented previously, and thus far in this paper, at pH 8, cAMP is required to induce a conformational change suggest that the response of cAMP receptor to cAMP prob- ably involves a conformational alteration resulting in an in- crease in the affinity of cAMP receptor for DNA. As an ex- 4.0 perimentally simple approach to this problem we have looked 3.0 3.0 0 E CU E .Eo _ 2.0 a) .-- + Trypsin CL 2.0 E 0~ x 1.0 + Subtilisin 1.0 Io~ ra 10-4 FIG. 4. Dependence of-cAMP concentration for inactivation of cAMP receptor by subtilisin and trypsin. The incubations con- pH tained (final volume, 0.25 ml): 40 mM Bis-Tris-propane buf- FIG. 2. Deoxypolymer binding by cAMP receptor as a func- fer (pH 8.0), 2.5 pug of cAMP receptor, and 0.5 pug of either sub- tion of pH and cAMP. The incubations contained (final volume, tilisin or trypsin. After addition of cAMP at the concentrations 0.25 ml): 40 mM Bis-Tris-propane buffer at the pH indicated, 2.5 indicated, the mixtures were incubated for 10 min at 370, after ug of cAMP receptor, 4.5 nmol of [3H]d(A-T) or [3H]d(I-C), which the following were added: 1 nmol of phenylmethanesul- and, where indicated, 4 X 10-4 M cAMP. The mixtures were in- fonylfluoride, 4.5 nmol of [3H]d(A-T), and 100 nmol of cAMP. cubated for 5 min at 37°. After addition of 0.75 ml of 50 mM After 5 min at 37°, 0.75 ml of 50 mM NaCl were added.
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