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Formation and properties of RNA-DNA complexes
J. Mol. Biol. (1964) 9, 125-142
Formation and Properties of RNA-DNA Complexes A. P.
NYGAARDt AND
B. D. 1IA.LL:j:
Department of Ohemistry, University of Illinois, Urbana, Illinois, U.S.A. (Received 27 NovemlJer 1963, and in revised form 16 March 1964) The rate of formation of complexes between '1'2 RNA and '1'2 DNA has been measured at various salt concentrations (0-2 to 1·5 M-KCl) and temperatures (50 to 85°C). With increasing temperature, the rate passes through a maximum which is higher the higher the salt concentration. In 0·5 M·KCl the optimum temperatureffi 67°0. The rate of reaction is proportional to both the RNA and DNA concentration. The apparent bimolecular constant for five different preparations of '1'2 RNA was 2 ml./fLg DNA/minute. or 10 l.jmole nucleotides/second (in 0'5 M-KCl, 67°0, pH 7·3). This rate is several orders of magnitude slower than that for the reaction between polyadenylic acid and polyuridylic acid. Differences and similarities between the two reactions are discussed. Annealing in the absence of UNA causes '1'2 DNA to lose its RNA-binding ability. The second-order rate constant for this process is approximately the same as that for the RNA-DNA reaction. Under the conditions used for their formation, RNA-DNA complexes are slowly destroyed. The fraction of complex breaking down in unit time increases as the concentration of RNA-DNA complex increases. . RNA-DNA complexes were broken down completely by RNase at low salt concentrations. The resistance to RNase increased with increasing salt concentration but was not complete under any condition tested. The complex was destroyed completely by DNase, The extent of complex formation was measured using an excess of either reactant. At least 77% of RNA formed after '1'2 infection of Escherichia coli is capable of becoming bound to T2 DNA. The binding capacity of '1'2 DNA is approximately 0·3 JLg RNA/fLg DNA.
1. Introduction Specific RNA-DNA complexes are formed when denatured DNA is annealed with RNA isolated from the same organism (Hall & Spiegelman, 1961). Similarly, RNA copied from DNA in vitro by RNA polymerase will form complexes with homologous DNA (Geiduschek. Nakamoto & WeiBB, 1961). It has generally been assumed that RNA-DNA complexes are held together by specific base-pair hydrogen bonds like those which join the two strands of DNA (Watson & Crick, 1953). This assumption is based mainly upon the requirement for complementarity in base composition (ARNA§ = TDNA , eto.) between the RNA and DNA which form the complex. There is as yet very little direct experimental evidence
t Present address: Department of Biochemistry, University of Bergen Medical School, Bergen, Norway. t Present address: Department of Genetics, University of Waahington, Seattle, WlIBhington, U.S.A. § Abbreviations used: At adenine; T, thymine; TeA, trichloroacetic acid; U, uracil. 125
126
A. P. NYGAARD AND B. D. HALL
concerning either the nature of the reaction between RNA and DNA or the properties of the complexes formed. This work is concerned with the kinetics of formation of RNA-DNA complexes and the composition and stability of the complexes under various conditions. Its purpose has been to define more clearly the nature of the reaction between RNA and DNA. Measurements of the rate and extent of RNA-DNA complex formation have been greatly aided by the availability of a quantitative assay for complex formation (Nygaard & Hall, 1963). Nitrocellulose membrane filters absorb RNA-DNA complexes, but allow free RNA to pass through. Measurement of the change with time in the quantity of RNA absorbable by nitrocellulose thus permits a direct observation of the rate of RNA-DNA complex formation.
2. Materials and Methods 32P-labeled T2 RNA RNA was pulse-labeled by addition of [32P]orthophosphate to T2-infected cultures of Escherichia coli B. The bacterial cultures were grown in modified C medium (Bautz & Hall, 1962) containing 2 X 10- 4 M-phosphate. At a cell titer of 3 X 108/ml., during logarithmic: growth, the bacteria were harvested by centrifugation. Mter resuspension in fresh, prewarmed C medium containing 10- 4 M-phosphate, growth was allowed to continue for 40 min at 30°C. Infection was begun by the addition of 5 to 10 T2 particlesfbacterium. The RNA formed during the latent period after T2 infection was labeled by the addition of [32P]orthophosphate to the infected culture. Incorporation of 32p was stopped by dumping the culture onto 0·4 vol, crushed, frozen 0·01 M-MgCI2, followed by centrifugation at O°C.Further operations, including RNA extraction, were carried out as described previously (Nygaard & Hall, 1963). The various RNA preparations will be denoted by giving the time of labeling within the latent period, the isotope, and the infecting phage (e.g. 15 to 19 min [32P]T2 RNA).
T2 DNA preparation and denaturation: (Nygaard & Hall, 1963) RNA-DNA complex formation Reaction mixtures were prepared by mixing RNA and DNA solutions at room temperature. When the volume was greater than 2 ml., the temperature was adjusted rapidly (in 30 to 40 sec) by immersing the tube in boiling water until the reaction temperature was attained. With smaller volumes, the tubes were placed directly in a constant-temperature bath at the reaction temperature. RNA-DNA complex was isolated and measured by absorption to nitrocellulose membrane filters. In kinetic experiments, the extent of reaction at a particular time was measured by quickly diluting a 10 to 100-p.I. portion of the reaction mixture into 15 ml. of 0·5 M-KCI, 0·01 M-tris pH 7,3, at room temperature. In the diluted state at this temperature, no further reaction occurs and RNA-DNA complex is stable for at least 24 hr; thus the sample can be filtered whenever time permits, and sampling can be done at short intervals (20 sec or less). The filter is soaked in the KCI solution for at least 10 min before use, and approximately 10 ml, of the same solution is passed through the filter before the sample is applied. Suction is provided by an aspirator, giving a vacuum of approximately 15 mm Hg, The filter is mounted on a stainless steel grid with a stainless steel cylinder placed on top. After absorption of the sample, the filter is washed with 60 ml, Of the KCl solution at room temperature, dried, placed in a vial with scintillating solution, and counted.
FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
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3. Results Effect o/temperature and Balt concentration upon RNA-DNA complex/ormation When saline solutions containing denatured T2 DNA and T2 RNA are heated above 50°0, RNA-DNA complex is produced: first at a constant rate, then at a decreasing rate (Fig. l(a)). The dependence of the initial rate upon temperature and salt concentration is shown in Fig. l(b). It is apparent that increasing the salt
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concentration (between 0·2 and 1·5 M-KOI) increases both the optimum temperature and the rate of complex form ation at the optimum temperature. For many of the experiments to follow, 0·5 M-KCl at 67°0 was chosen as a standard condition.
Dependence 0/ reaction rate upon RNA and DNA concentration Studies of the initial reaction velocity over a 96-fold range of RNA concentration and an 18-fold range of DNA concentration in 0·5 M-KOI are recorded in Figs. 2, 3(a) and (b). The rate of complex formation is shown to be proportional to the first power of both RNA and DNA concentrations. A similar proportionality of reaction rate has been obtained in 1l\I-KCl at the same temperature (67°0) . The reaction rate was measured with five different RNA preparations, pulselabeled with 32p at different times after T2 infection (Fig . 4). For each, at a given DNA concentration the specific rate at which RNA was bound was independent of RNA concentration, as in Fig. 3. Thus the data for each series of RNA concentrations with a given RNA preparation, at a particular DNA concentration, is represented by a point in Fig. 4.
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FIG. 2. Rate of complllXformation as a function of DNA concentration. Heat-denetured T2 DNA at various concentrations. 14·1 p.g{ml. of 15 to 19 min 32P-labeled T2 RNA, 8900 cts{min{p.g; 0·5 M-KCl at 67°C. Inset: Initial rates plotted ver8U8 DNA concentration.
The reasonable adherence of the points to a single straight line shows that the rate is proportional to both RNA and DNA concentrations, with the same second-order constant for the various RNA preparations. An apparent value of 1·4 X 10- 3 ml.jp.g DNA'min for this rate constant is obtained from the ratio of ordinate/abscissa in Fig. 4. Actually the ordinate of Fig. 4 is: . [32PJRNA in complex . Increase of I CA bl 32p per rom. tota T -ppt e. Of the total TCA-precipitable [32PJRNA of the various preparations, approximately 70% was capable of reaction with excess T2 DNA (see legend to Fig. 4). Since the concentration of reactive RNA has been over-estimated in Fig. 4, the apparent secondorder constant is too low. The corrected value is 2·0 X 10- 3 ml./p.g DNA·min.
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FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
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Denaturation and renaturation of DNA In contrast to denatured T2 DNA, native T2 DNA does not bind T2 RNA when annealed with it (Hall & Spiegelman, 1961; Nygaard & Hall, 1963). To explore further the relation between RNA-DNA complex formation and the thermal transition undergone by DNA, we might ask: How vigorously must native T2 DNA be heated in order to render it fully reactive toward T2 RNA1 Heating T2 DNA for two to three seconds at 90°0 after warming from 25 to 90°0 in a 45-BCcond period proved sufficient to achieve activation for complex formation (Fig. 5).
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FIG. 6. Effect of DNA denaturation procedure upon complex formation . Conditions for complex formation: 22 p.g/ml. T2 DNA; n'7 p.g/ml. 15 to 19 min 32P-Iabeled T2 RNA, 1720 cts/min/p.g; 0·5 H-KCI at 67°C. DNA denaturation procedure: ( - x - x - ) boiled for 15 min at 27 p.g/ml. DNA in 0·01 H-tris0·06 H-KCl and cooled rapidly; (-0-0-) DNA solution in 0·01 H-tris-0'06 H-KCI, at 27 JLg/ml., was heated from 20 to 90°C in 45 sec and then poured directly into the RNA solution (at 20°C) to begin. the reaction.
Because of the similar optimum conditions for DNA renaturation and RNA-DNA complex formation (Fig. 1(b), Marmur & Doty, 1961), DNA might be expected to lose ita reactivity during the course of the RNA-DNA reaction. The effect of renaturation upon the reactivity of DNA was studied by annealing denatured T2 DNA under the reaction conditions, but without RNA, and then adding RNA after various times. The results (Fig. 6) show sufficient renaturation at 24 and 6 fLgfml. DNA to reduce
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FIG. 6. Rate of complexing RNA after annealing DNA. (a) Conditions for annealing DNA: denatured DNA, 24 p.g/mI., was incubated in 0·5 M-RCI-O'OI M-tris, pH 7·3, at 67°C for 10 or 20 min. Conditions for complex formation: heat-denatured T4 DNA, 24 p.g/mI., in all experiments; T4 RNA from uniformly 32P-Iabeled E. coli, 12·2 p.g/mI., 22,000 cts/min/p.g; 0·5 M.RCI, 67°C. Sample: 1·0 mI. (b) Conditions for annealing DNA: denatured DNA, 6 p.g/mI., was incubated in 0·5 M·RCI0·01 M-tris, pH 7·3 at 67°C for 20 min. Conditions for complex formation: heat-denatured T4 DNA, 6 p.g/mi., in both experiments; 3 to 10 min 32P-Iabeled T4 RNA, 13·6 p.g/mI., 488 cts/min/p.g; 0·5 M.RCI, 67°C. Sample: 1·0 mI. (c) Conditions for annealing DNA: denatured DNA, 2 p.g/mI., was incubated in 0·5 M-RCI0·01 M-tris, pH 7'3, at 67°C for 20 min. Conditions for complex formation: heat-denatured T4 DNA, 2 p.g/mI., in both experiments; 3 to 10 min 32P-Iabeled T4 RNA, 15·4 p.g/mi., 488 cts/ min/p.g; 0·5 M.RCI, 67°C. Sample: 3·0 mI. ::::, 1500..----,-----,----,----" :l..
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FIG. 7. Rate and extent of complexing RNA after annealing T2 DNA. Condibions for annealing DNA: denatured [3H]DNA, 2·0 p.g/mI., 4000 cts/min{p.g incubated in 1·5 M-RCI-O'Ol M.tris, pH 7,3, at 76°C. (a) Conditions for complex formation: 0 to 20 min 32P-Iabeled T2 RNA, 162 p.g/mI., 1750 cts/ min{,..g was incubated with I ,..g/mI. DNA in I M-KOI at 67°0. (-e-e-) No annealing of DNA; (-0-0-) annealing of DNA for 1 hr; ( - x - x - ) annealing of DNA for 3 hr. (b) Remaining ability to bind RNA a8 meaeured by: (-e-e-) initial rate (Fig. 7(a)); ( - x - x-) extent of the reaction (Fig. 7(a». The curves are calculated for a simple second-order reaction.
FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
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the rate of complex formation appreciably, but no measurable renaturation in 20 minutes at 2 p-gJml. DNA. These observations are in agreement with the notion that DNA renaturation is a bimolecular reaction. The second-order rate constant derived here is approximately 2 X 10- 3 ml.!p,g DNA ·minute. Thus, under the same conditions, the DNA-DNA and DNA-RNA reactions proceed at the same rate. At 2 p-g!ml. DNA, renaturation can be made to occur at an appreciable rate by raising the temperature to 76°0 and the KCl concentration to 1·5 M (Fig. 7). It is seen that DNA renaturation decreases both the initial rate and final extent of the RNA-DNA reaction, although not to the same degree. The decrease in observable RNA-DNA complex formation is not caused by decreased absorption of DNA by nitrocellulose. Throughout the renaturation and subsequent annealing with RNA, the DNA continues to be completely absorbed. Although DNA renaturation can significantly affect the final level of RNA-DNA complex formation in many experiments, the initial rate can be measured without interference. By removing samples at 20 to 30 second intervals, it has been possible to measure the initial rate of complex formation at DNA concentrations as high as 50 p,gJml. Decomposition of RNA-DNA complex
Mter prolonged annealing, a spontaneous disappearance of RNA-DNA complex is noted (Fig. 8). The reason for this remains obscure; however, several conceivable causes have been eliminated. (1) Annealing may convert RNA-DNA complex to a physical state in which it is not absorbed by nitrocellulose. Experimentally, it was found that, following decomposition of RNA-DNA complex to the extent of 40%, all the DNA (SH-labeled) is still retained by nitrocellulose. (2) The RNA of the RNA-DNA complex may be hydrolysed or otherwise damaged at the elevated temperature. Experimentally, RNA was found to be completely stable for six hours at 67°0, as measured by TOA precipitation. Furthermore, there is no decrease of the rate with which RNA-DNA complex is formed when RNA is boiled for five minutes before addition of DNA or is preincubated for two hours at 67°0. (3) The RNA-DNA complex which is formed at high concentrations of RNA may contain imperfections, such as free ends of RNA molecules, resulting from multiple occupancy of some DNA sites. What appears to be decomposition may in fact be a rearrangement of imperfect complexes to more nearly perfect ones, with a concomitant loss of RNA. The free RNA ends and other imperfections should be labile to pancreatic ribonuclease (Yankofsky & Spiegelman, 1962). The results of RNase experiments (see below) provide no evidence that imperfect complexes are formed. The disappearance of RNA-DNA complex becomes more rapid with increasing concentration. The relative rate of breakdown is slower for complexes formed at low DNA and RNA concentration (Figs 8 and 9). In addition, breakdown can be slowed considerably by dilution of preformed RNA-DNA complex (Fig. 9). Thus it appears that the decomposition is not unimolecular, but involves reaction between an RNADNA complex and some other entity, either a free DNA strand, an RNA strand, or another RNA-DNA complex.
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FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
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Titration of RNA with exce88 DNA To estimate the degree of completion of the reaction of RNA with DNA, it is first of all necessary to measure the total amount of reactive RNA present. That is, given 1 mg of pulse-labeled RNA from T2-infected bacteria, having a specific activity of x countsjminjmg, what fraction of x is capable of complexing with T2 DNA1 This fraction is expected to be near 1, since apparently only T2-specific RNA is made following T2 infection of E. coli (Volkin & Astrachan, 1956). However, a small contamination of the culture with uninfected cells can contribute a disproportionate amount of labeled bacterial RNA. The titration of T2 RNA labeled with 32p between 15 and 19 minutes, is shown in Fig. 10. To achieve a sufficient excess of DNA and yet avoid excessive renaturation of DNA, the latter was added in two successive increments. The reaction mixture was filtered through nitrocellulose before the second addition in order to remove DNA and RNA-DNA complex previously present. Of the [32P]RNA originally present, 60% was complexed by DNA in the first reaction period and 12% in the second. Since some reactive RNA may have remained at the end, at least 72% of the [32P]RNA is capable of forming complexes with T2 DNA.
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The results also show that the various T2 RNA molecules present react with T2 RNA at a similar rate. If they did not, the specific rate of the first 60% of the RNA to react would exceed that of the RNA reacting later. The observed rate of complex formation after the second addition of DNA is 0·16 of that after the first addition. This is in good agreement with the ratio of reactive RNA concentrations for the two (0'12/0'72 = 0,17).
FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
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Stoicheiometry of RNA-DNA complexes Figure 11 shows the result obtained when a given amount of DNA (22 p,g) is annealed with varying quantities of RNA. At the higher levels, such a great excess of RNA is present that the reaction is 75% complete within two minutes; hence DNA renaturation does not affect these points. The horizontal dashed line represents the total capacity of T2 DNA to bind the [32PJRNA preparation. The nearly vertical dashed line marks the total available RNA reactant at each input RNA level. (Titration of the RNA preparation with excess DNA showed that 80% of the [32PJRNA could be complexed by T2 DNA.) The RNA/DNA ratio at which the dashed lines intersect thus specifies the composition of RNA-DNA complex which is saturated with RNA. The results show that each
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microgram of T2 DNA can bind the amount of T2-specific RNA present in 5·5 p,g of RNA extracted from T2-infected bacteria. Titration of RNA from Uniformly 32p_ labeled T2-infected E. coli by the procedure of Fig. 10 showed that 5% of the total RNA could be bound by T2 D~A. If 5% of the RNA preparation used to saturate DNA (Fig. 11) is T2-specific RNA, then 0·3 p,g RNA is bound by each microgram of DNA. At the equivalence point (RNA/DNA = 5,5) the binding of RNA is only 37% complete (Fig. 11). It is likely that the reaction is incomplete because of DNA renaturation (which occurs at the same rate as RNA-DNA complex formation) and/or decomposition of the complex.
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Stability of RNA-DNA complexes to various agents
Nuclease» In solutions oflow ionic strength, RNA-DNA complex is completely destroyed by RNase (Fig . 12). In solutions 0·2 M or higher in KCI concentration, an appreciable
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fraction of the complex is stable to RNase. Complexes formed with varying concentrations of the reactants have been tested in 0·5 M-KCl. The RNase.stable fraction varied from 50 to 80%. The stabilization apparently results from the binding of RNA to DNA , since under the same assay conditions, in both 0·2 M and 0·5 M-KCl, free RNA was 95% degraded to fragments soluble in TCA. The fraction of RNase-labile complex remains constant both during the formation of the complex (Fig. 13) and during its spont aneous decomposition. Upon treatment with 0·55 p.gJml. of pancreatic DNase at 30°C, RNA-DNA complex is completely destroyed. The assay used was ab sorption on nitrocellulose of complex containing labeled RNA.
FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
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Dilution Measurement of the rate of dissociation of RNA-DNA complex is complicated by the spontaneous decomposition which occurs at high concentration. Attempts to measure dissociation at 67°C, either by simple dilution of preformed complex or by exchange with a 20-fold excess of free RNA, have shown that the dissociation of RNA-DNA complex is extremely slow. No definite exchange between free and bound RNA could be observed after four hours incubation at 67°C. A 4O-fold dilution of complex in 0·5 M-RCI, followed by 12 hours incubation at 67°C, produced no dissociation.
HeaJ,ing RNA-DNA complex formed at 67°C in 0·5 M-KCI dissociates when it is heated above this temperature. A constant level of dissociation is quickly attained at each temperature (Fig. 14). At 83°C, dissociation is 50% complete. Extremes of pH
In 0'5 M-RCI at 25°C, RNA-DNA complex is stable to 30 minutes treatment at any pH between 1 and 10. Dissociation was observed above pH 10.
140
A. P. NYGAARD AND B. D. HALL
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4. Discussion Nature of the reaction product The hypothesis that RNA-DNA complex formation involves the union of single RNA and DNA strands is strongly supported by our kinetic data. The rate at which RNA becomes bound to DNA is proportional to the product (RNA concentration) X (DNA concentration) over a 1O,OOO.fold range. The quantity of RNA bound by a given amount of T2 DNA is limited, as it must be for complexes having a definite structure. The maximum amount bound, approximately 0·3 p,g RNAIp,g T2 DNA , is consistent with the complex being one-strand RNA and one-strand DNA, provided certain regions of the DNA do not participate in the reaction. (Previous experiments (Hall, Green, Nygaard & Boezi, 1963) have shown that only about 50% of the polynucleotide chains of denatured T2 DNA are capable of binding T2 RNA.) The resistance of RNA-DNA complexes to digestion by RNase is considerably less than that previously estimated (Yankofsky & Spiegelman, 1962). The use of resistance to RNase as a measure of specific RNA-DNA complex formation may thus lead to under-estimation of the extent of reaction. Significance of kinetic constants Measurement of the second-order rate constant, and the effect of temperature and salt concentration upon the rate, permit a comparison between RNA-DNA complex formation and the related polynucleotide reactions: poly A poly U (Ross & Sturtevant, 1962) and DNA renaturation (l\:Iarmur & Doty, 1961).
+
FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
141
All three reactions exhibit a maximum in the rate versus temperature curve. For poly A + poly U the maximum rate occurs 40°C below T m- the melting temperature; for DNA renaturation, 25°C below T m; and for RNA + DNA, 15 to 20°C below T m (Figs l(b) and 14). The apparent second-order rate constants are: (a) for poly A + poly U, 1·5 X 106 l.fmole sec at 34°C in 0·5 M-NaCI; (b) for renaturation of Diplococcus pneumoniae DNA, 21.jmole sec at 67°C in 0·3 M.NaCI, 0·03 M-sodium citrate; (c) for T2 RNA + T2 DNA, 10 l.jmole sec at 67°0 in 0·5 M-KCI-Q·Ol M-tris, pH 7·3 (if 50% of the T2 DNA is considered incapable of binding RNA, this becomes 20 l.jmole sec). In all three cases the units of concentration used are molarity of nucleotides and not the molar concentration of nucleic acid molecules. It is evident that, under equivalent conditions, DNA renaturation and RNA-DNA complex formation occur at similar rates, but that both reactions are several orders of magnitude slower than the reaction of poly A with poly U. The slowness of the RNA-DNA and DNA-DNA reactions appears to be a consequence of the requirement for bringing together complementary sequences. This would cause the reactions to be slower than that of the simple homopolynucleotides for at least two reasons. (I) They would be slower because the rate of reaction of any given sequence is governed not by the over-all DNA concentration, but by the much lower concentration of the one complementary sequence. If this were the only reason for the slow apparent rate, one might conclude that the T2 RNA-T2 DNA reaction consists of nearly 105 separate and independent reactions. (2) In addition, the RNA-DNA and DNA-DNA reactions would be slower because of the many accidental sequence pairings which possess some homology and therefore can persist long enough to impede the exploration needed to arrive at the "correct" base-pairing. The dependence on temperature of the rate at sub-optimal temperatures may be related to accidental pairing. The activation energy calculated for the region of maximum positive slope is: 3·5 kcal.jmole for poly A + poly U (in 0·5 M-NaOI), 20 koal.jmole for pneumococcal DNA renaturation (in 0·3 M·NaOI-Q·03 M-sodium citrate), and 23 kcal.jmole for the RNA-DNA reaction (in 0·5 M-KCI-Q·Ol M-tris, pH 7,3). Thus the two reactions which involve matching of complex nucleotide sequences require more energetic conditions, apparently in order to overcome some barrier to reaction. This barrier may be the restriction of conformational freedom caused by accidental pairing of the polynucleotide chains. We gratefully acknowledge the rapidity and precision of Kathleen Kliewer in helping us to obtain rapid kinetic data. This investigation was supported by a PHS research grant (A 3086) from the National Institute of Arthritis and Metabolic Diseases, Public Health Service. One of us (A. P. N.) would like to express his gratitude to Dr. H. O. Halvorsen for making possible his tenure in the Department of Microbiology of the University of Illinois. REFERENCES Bautz, E. K. F. & Hall, B. D. (1962). Proc, Nat. Acad. Sci., Wash. 48, 400. Geiduschek, E. P., Nakamoto, T. & Weiss, S. B. (1961). Proc, Nat. Acad. Sci., Wash. 47, 1405.
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Hall, B. D. & Spiegelman, S. (1961). Proc, Nat. Acad. Sci., Wash. 47, 137. Ha.ll, B. D., Green, M. H., Nygaard, A. P. & Boezi, J. (1963). 0014 Spr. Harb . Symp. Quant. B iol. 28, 201. . Marmur, J. & Doty, P. (1961). J. Mol. Biol. 3, 585. Nygaard, A. P. & Hall, B. D. (1963). Biochem, Biophys. Res. Oomm. 12, 98. Ross, P. D. & Sturtevant, J. M. (1962) . J. Amer. ahem. Soc. 84, 4503. Volkin, E. & Astrachan, L. (1956), Virology, 2, 149. Wa.tson, J . D. & Crick, F. H. C. (1953). Nature, 171, 737. Yankofsky, S. A. & Spiegelman, S. (1962). Proc. Nat. Acad. WaBh.48, 1069.
ss;
Formation and Properties of RNA-DNA Complexes A. P.
NYGAARDt AND
B. D. 1IA.LL:j:
Department of Ohemistry, University of Illinois, Urbana, Illinois, U.S.A. (Received 27 NovemlJer 1963, and in revised form 16 March 1964) The rate of formation of complexes between '1'2 RNA and '1'2 DNA has been measured at various salt concentrations (0-2 to 1·5 M-KCl) and temperatures (50 to 85°C). With increasing temperature, the rate passes through a maximum which is higher the higher the salt concentration. In 0·5 M·KCl the optimum temperatureffi 67°0. The rate of reaction is proportional to both the RNA and DNA concentration. The apparent bimolecular constant for five different preparations of '1'2 RNA was 2 ml./fLg DNA/minute. or 10 l.jmole nucleotides/second (in 0'5 M-KCl, 67°0, pH 7·3). This rate is several orders of magnitude slower than that for the reaction between polyadenylic acid and polyuridylic acid. Differences and similarities between the two reactions are discussed. Annealing in the absence of UNA causes '1'2 DNA to lose its RNA-binding ability. The second-order rate constant for this process is approximately the same as that for the RNA-DNA reaction. Under the conditions used for their formation, RNA-DNA complexes are slowly destroyed. The fraction of complex breaking down in unit time increases as the concentration of RNA-DNA complex increases. . RNA-DNA complexes were broken down completely by RNase at low salt concentrations. The resistance to RNase increased with increasing salt concentration but was not complete under any condition tested. The complex was destroyed completely by DNase, The extent of complex formation was measured using an excess of either reactant. At least 77% of RNA formed after '1'2 infection of Escherichia coli is capable of becoming bound to T2 DNA. The binding capacity of '1'2 DNA is approximately 0·3 JLg RNA/fLg DNA.
1. Introduction Specific RNA-DNA complexes are formed when denatured DNA is annealed with RNA isolated from the same organism (Hall & Spiegelman, 1961). Similarly, RNA copied from DNA in vitro by RNA polymerase will form complexes with homologous DNA (Geiduschek. Nakamoto & WeiBB, 1961). It has generally been assumed that RNA-DNA complexes are held together by specific base-pair hydrogen bonds like those which join the two strands of DNA (Watson & Crick, 1953). This assumption is based mainly upon the requirement for complementarity in base composition (ARNA§ = TDNA , eto.) between the RNA and DNA which form the complex. There is as yet very little direct experimental evidence
t Present address: Department of Biochemistry, University of Bergen Medical School, Bergen, Norway. t Present address: Department of Genetics, University of Waahington, Seattle, WlIBhington, U.S.A. § Abbreviations used: At adenine; T, thymine; TeA, trichloroacetic acid; U, uracil. 125
126
A. P. NYGAARD AND B. D. HALL
concerning either the nature of the reaction between RNA and DNA or the properties of the complexes formed. This work is concerned with the kinetics of formation of RNA-DNA complexes and the composition and stability of the complexes under various conditions. Its purpose has been to define more clearly the nature of the reaction between RNA and DNA. Measurements of the rate and extent of RNA-DNA complex formation have been greatly aided by the availability of a quantitative assay for complex formation (Nygaard & Hall, 1963). Nitrocellulose membrane filters absorb RNA-DNA complexes, but allow free RNA to pass through. Measurement of the change with time in the quantity of RNA absorbable by nitrocellulose thus permits a direct observation of the rate of RNA-DNA complex formation.
2. Materials and Methods 32P-labeled T2 RNA RNA was pulse-labeled by addition of [32P]orthophosphate to T2-infected cultures of Escherichia coli B. The bacterial cultures were grown in modified C medium (Bautz & Hall, 1962) containing 2 X 10- 4 M-phosphate. At a cell titer of 3 X 108/ml., during logarithmic: growth, the bacteria were harvested by centrifugation. Mter resuspension in fresh, prewarmed C medium containing 10- 4 M-phosphate, growth was allowed to continue for 40 min at 30°C. Infection was begun by the addition of 5 to 10 T2 particlesfbacterium. The RNA formed during the latent period after T2 infection was labeled by the addition of [32P]orthophosphate to the infected culture. Incorporation of 32p was stopped by dumping the culture onto 0·4 vol, crushed, frozen 0·01 M-MgCI2, followed by centrifugation at O°C.Further operations, including RNA extraction, were carried out as described previously (Nygaard & Hall, 1963). The various RNA preparations will be denoted by giving the time of labeling within the latent period, the isotope, and the infecting phage (e.g. 15 to 19 min [32P]T2 RNA).
T2 DNA preparation and denaturation: (Nygaard & Hall, 1963) RNA-DNA complex formation Reaction mixtures were prepared by mixing RNA and DNA solutions at room temperature. When the volume was greater than 2 ml., the temperature was adjusted rapidly (in 30 to 40 sec) by immersing the tube in boiling water until the reaction temperature was attained. With smaller volumes, the tubes were placed directly in a constant-temperature bath at the reaction temperature. RNA-DNA complex was isolated and measured by absorption to nitrocellulose membrane filters. In kinetic experiments, the extent of reaction at a particular time was measured by quickly diluting a 10 to 100-p.I. portion of the reaction mixture into 15 ml. of 0·5 M-KCI, 0·01 M-tris pH 7,3, at room temperature. In the diluted state at this temperature, no further reaction occurs and RNA-DNA complex is stable for at least 24 hr; thus the sample can be filtered whenever time permits, and sampling can be done at short intervals (20 sec or less). The filter is soaked in the KCI solution for at least 10 min before use, and approximately 10 ml, of the same solution is passed through the filter before the sample is applied. Suction is provided by an aspirator, giving a vacuum of approximately 15 mm Hg, The filter is mounted on a stainless steel grid with a stainless steel cylinder placed on top. After absorption of the sample, the filter is washed with 60 ml, Of the KCl solution at room temperature, dried, placed in a vial with scintillating solution, and counted.
FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
127
3. Results Effect o/temperature and Balt concentration upon RNA-DNA complex/ormation When saline solutions containing denatured T2 DNA and T2 RNA are heated above 50°0, RNA-DNA complex is produced: first at a constant rate, then at a decreasing rate (Fig. l(a)). The dependence of the initial rate upon temperature and salt concentration is shown in Fig. l(b). It is apparent that increasing the salt
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concentration (between 0·2 and 1·5 M-KOI) increases both the optimum temperature and the rate of complex form ation at the optimum temperature. For many of the experiments to follow, 0·5 M-KCl at 67°0 was chosen as a standard condition.
Dependence 0/ reaction rate upon RNA and DNA concentration Studies of the initial reaction velocity over a 96-fold range of RNA concentration and an 18-fold range of DNA concentration in 0·5 M-KOI are recorded in Figs. 2, 3(a) and (b). The rate of complex formation is shown to be proportional to the first power of both RNA and DNA concentrations. A similar proportionality of reaction rate has been obtained in 1l\I-KCl at the same temperature (67°0) . The reaction rate was measured with five different RNA preparations, pulselabeled with 32p at different times after T2 infection (Fig . 4). For each, at a given DNA concentration the specific rate at which RNA was bound was independent of RNA concentration, as in Fig. 3. Thus the data for each series of RNA concentrations with a given RNA preparation, at a particular DNA concentration, is represented by a point in Fig. 4.
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The reasonable adherence of the points to a single straight line shows that the rate is proportional to both RNA and DNA concentrations, with the same second-order constant for the various RNA preparations. An apparent value of 1·4 X 10- 3 ml.jp.g DNA'min for this rate constant is obtained from the ratio of ordinate/abscissa in Fig. 4. Actually the ordinate of Fig. 4 is: . [32PJRNA in complex . Increase of I CA bl 32p per rom. tota T -ppt e. Of the total TCA-precipitable [32PJRNA of the various preparations, approximately 70% was capable of reaction with excess T2 DNA (see legend to Fig. 4). Since the concentration of reactive RNA has been over-estimated in Fig. 4, the apparent secondorder constant is too low. The corrected value is 2·0 X 10- 3 ml./p.g DNA·min.
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FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
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Denaturation and renaturation of DNA In contrast to denatured T2 DNA, native T2 DNA does not bind T2 RNA when annealed with it (Hall & Spiegelman, 1961; Nygaard & Hall, 1963). To explore further the relation between RNA-DNA complex formation and the thermal transition undergone by DNA, we might ask: How vigorously must native T2 DNA be heated in order to render it fully reactive toward T2 RNA1 Heating T2 DNA for two to three seconds at 90°0 after warming from 25 to 90°0 in a 45-BCcond period proved sufficient to achieve activation for complex formation (Fig. 5).
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Because of the similar optimum conditions for DNA renaturation and RNA-DNA complex formation (Fig. 1(b), Marmur & Doty, 1961), DNA might be expected to lose ita reactivity during the course of the RNA-DNA reaction. The effect of renaturation upon the reactivity of DNA was studied by annealing denatured T2 DNA under the reaction conditions, but without RNA, and then adding RNA after various times. The results (Fig. 6) show sufficient renaturation at 24 and 6 fLgfml. DNA to reduce
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FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
133
the rate of complex formation appreciably, but no measurable renaturation in 20 minutes at 2 p-gJml. DNA. These observations are in agreement with the notion that DNA renaturation is a bimolecular reaction. The second-order rate constant derived here is approximately 2 X 10- 3 ml.!p,g DNA ·minute. Thus, under the same conditions, the DNA-DNA and DNA-RNA reactions proceed at the same rate. At 2 p-g!ml. DNA, renaturation can be made to occur at an appreciable rate by raising the temperature to 76°0 and the KCl concentration to 1·5 M (Fig. 7). It is seen that DNA renaturation decreases both the initial rate and final extent of the RNA-DNA reaction, although not to the same degree. The decrease in observable RNA-DNA complex formation is not caused by decreased absorption of DNA by nitrocellulose. Throughout the renaturation and subsequent annealing with RNA, the DNA continues to be completely absorbed. Although DNA renaturation can significantly affect the final level of RNA-DNA complex formation in many experiments, the initial rate can be measured without interference. By removing samples at 20 to 30 second intervals, it has been possible to measure the initial rate of complex formation at DNA concentrations as high as 50 p,gJml. Decomposition of RNA-DNA complex
Mter prolonged annealing, a spontaneous disappearance of RNA-DNA complex is noted (Fig. 8). The reason for this remains obscure; however, several conceivable causes have been eliminated. (1) Annealing may convert RNA-DNA complex to a physical state in which it is not absorbed by nitrocellulose. Experimentally, it was found that, following decomposition of RNA-DNA complex to the extent of 40%, all the DNA (SH-labeled) is still retained by nitrocellulose. (2) The RNA of the RNA-DNA complex may be hydrolysed or otherwise damaged at the elevated temperature. Experimentally, RNA was found to be completely stable for six hours at 67°0, as measured by TOA precipitation. Furthermore, there is no decrease of the rate with which RNA-DNA complex is formed when RNA is boiled for five minutes before addition of DNA or is preincubated for two hours at 67°0. (3) The RNA-DNA complex which is formed at high concentrations of RNA may contain imperfections, such as free ends of RNA molecules, resulting from multiple occupancy of some DNA sites. What appears to be decomposition may in fact be a rearrangement of imperfect complexes to more nearly perfect ones, with a concomitant loss of RNA. The free RNA ends and other imperfections should be labile to pancreatic ribonuclease (Yankofsky & Spiegelman, 1962). The results of RNase experiments (see below) provide no evidence that imperfect complexes are formed. The disappearance of RNA-DNA complex becomes more rapid with increasing concentration. The relative rate of breakdown is slower for complexes formed at low DNA and RNA concentration (Figs 8 and 9). In addition, breakdown can be slowed considerably by dilution of preformed RNA-DNA complex (Fig. 9). Thus it appears that the decomposition is not unimolecular, but involves reaction between an RNADNA complex and some other entity, either a free DNA strand, an RNA strand, or another RNA-DNA complex.
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FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
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Titration of RNA with exce88 DNA To estimate the degree of completion of the reaction of RNA with DNA, it is first of all necessary to measure the total amount of reactive RNA present. That is, given 1 mg of pulse-labeled RNA from T2-infected bacteria, having a specific activity of x countsjminjmg, what fraction of x is capable of complexing with T2 DNA1 This fraction is expected to be near 1, since apparently only T2-specific RNA is made following T2 infection of E. coli (Volkin & Astrachan, 1956). However, a small contamination of the culture with uninfected cells can contribute a disproportionate amount of labeled bacterial RNA. The titration of T2 RNA labeled with 32p between 15 and 19 minutes, is shown in Fig. 10. To achieve a sufficient excess of DNA and yet avoid excessive renaturation of DNA, the latter was added in two successive increments. The reaction mixture was filtered through nitrocellulose before the second addition in order to remove DNA and RNA-DNA complex previously present. Of the [32P]RNA originally present, 60% was complexed by DNA in the first reaction period and 12% in the second. Since some reactive RNA may have remained at the end, at least 72% of the [32P]RNA is capable of forming complexes with T2 DNA.
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FIG. 10. DNA.binding capacity of RNA. Reaction I (-X-X-): RNA (14 f£g/ml.) was allowed to react with excess DNA (25 f£g/ml.) for 70 min. Conditione: 1·2 ml. heat- denatured DNA (83 f£g/ml.); 0·1 ml. 15 to 19 min, 32P·labeled T2 RNA, 563 f£g/ml., 12,700 cts/min/f£g; 2·46 ml. 0·5 M.KCI; 0·24 ml. 3·0 M·KCl. Incubated for 70 min at 67°0, then cooled and filtered through nitrocellulose membrane filters, I ml./filter. Reaction II ( - e - e - ) : to 1·28 ml. of the filtrate from reaction I, 0·60 ml. DNA (83 f£g/ml.) and 0·12 ml. 3·0 M·KCI were added. Reaction at 67°C was then resumed.
The results also show that the various T2 RNA molecules present react with T2 RNA at a similar rate. If they did not, the specific rate of the first 60% of the RNA to react would exceed that of the RNA reacting later. The observed rate of complex formation after the second addition of DNA is 0·16 of that after the first addition. This is in good agreement with the ratio of reactive RNA concentrations for the two (0'12/0'72 = 0,17).
FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
137
Stoicheiometry of RNA-DNA complexes Figure 11 shows the result obtained when a given amount of DNA (22 p,g) is annealed with varying quantities of RNA. At the higher levels, such a great excess of RNA is present that the reaction is 75% complete within two minutes; hence DNA renaturation does not affect these points. The horizontal dashed line represents the total capacity of T2 DNA to bind the [32PJRNA preparation. The nearly vertical dashed line marks the total available RNA reactant at each input RNA level. (Titration of the RNA preparation with excess DNA showed that 80% of the [32PJRNA could be complexed by T2 DNA.) The RNA/DNA ratio at which the dashed lines intersect thus specifies the composition of RNA-DNA complex which is saturated with RNA. The results show that each
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microgram of T2 DNA can bind the amount of T2-specific RNA present in 5·5 p,g of RNA extracted from T2-infected bacteria. Titration of RNA from Uniformly 32p_ labeled T2-infected E. coli by the procedure of Fig. 10 showed that 5% of the total RNA could be bound by T2 D~A. If 5% of the RNA preparation used to saturate DNA (Fig. 11) is T2-specific RNA, then 0·3 p,g RNA is bound by each microgram of DNA. At the equivalence point (RNA/DNA = 5,5) the binding of RNA is only 37% complete (Fig. 11). It is likely that the reaction is incomplete because of DNA renaturation (which occurs at the same rate as RNA-DNA complex formation) and/or decomposition of the complex.
138
A. P. NYGAARD AND B. D. HALL
Stability of RNA-DNA complexes to various agents
Nuclease» In solutions oflow ionic strength, RNA-DNA complex is completely destroyed by RNase (Fig . 12). In solutions 0·2 M or higher in KCI concentration, an appreciable
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fraction of the complex is stable to RNase. Complexes formed with varying concentrations of the reactants have been tested in 0·5 M-KCl. The RNase.stable fraction varied from 50 to 80%. The stabilization apparently results from the binding of RNA to DNA , since under the same assay conditions, in both 0·2 M and 0·5 M-KCl, free RNA was 95% degraded to fragments soluble in TCA. The fraction of RNase-labile complex remains constant both during the formation of the complex (Fig. 13) and during its spont aneous decomposition. Upon treatment with 0·55 p.gJml. of pancreatic DNase at 30°C, RNA-DNA complex is completely destroyed. The assay used was ab sorption on nitrocellulose of complex containing labeled RNA.
FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
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Dilution Measurement of the rate of dissociation of RNA-DNA complex is complicated by the spontaneous decomposition which occurs at high concentration. Attempts to measure dissociation at 67°C, either by simple dilution of preformed complex or by exchange with a 20-fold excess of free RNA, have shown that the dissociation of RNA-DNA complex is extremely slow. No definite exchange between free and bound RNA could be observed after four hours incubation at 67°C. A 4O-fold dilution of complex in 0·5 M-RCI, followed by 12 hours incubation at 67°C, produced no dissociation.
HeaJ,ing RNA-DNA complex formed at 67°C in 0·5 M-KCI dissociates when it is heated above this temperature. A constant level of dissociation is quickly attained at each temperature (Fig. 14). At 83°C, dissociation is 50% complete. Extremes of pH
In 0'5 M-RCI at 25°C, RNA-DNA complex is stable to 30 minutes treatment at any pH between 1 and 10. Dissociation was observed above pH 10.
140
A. P. NYGAARD AND B. D. HALL
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4. Discussion Nature of the reaction product The hypothesis that RNA-DNA complex formation involves the union of single RNA and DNA strands is strongly supported by our kinetic data. The rate at which RNA becomes bound to DNA is proportional to the product (RNA concentration) X (DNA concentration) over a 1O,OOO.fold range. The quantity of RNA bound by a given amount of T2 DNA is limited, as it must be for complexes having a definite structure. The maximum amount bound, approximately 0·3 p,g RNAIp,g T2 DNA , is consistent with the complex being one-strand RNA and one-strand DNA, provided certain regions of the DNA do not participate in the reaction. (Previous experiments (Hall, Green, Nygaard & Boezi, 1963) have shown that only about 50% of the polynucleotide chains of denatured T2 DNA are capable of binding T2 RNA.) The resistance of RNA-DNA complexes to digestion by RNase is considerably less than that previously estimated (Yankofsky & Spiegelman, 1962). The use of resistance to RNase as a measure of specific RNA-DNA complex formation may thus lead to under-estimation of the extent of reaction. Significance of kinetic constants Measurement of the second-order rate constant, and the effect of temperature and salt concentration upon the rate, permit a comparison between RNA-DNA complex formation and the related polynucleotide reactions: poly A poly U (Ross & Sturtevant, 1962) and DNA renaturation (l\:Iarmur & Doty, 1961).
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FORMATION AND PROPERTIES OF RNA-DNA COMPLEXES
141
All three reactions exhibit a maximum in the rate versus temperature curve. For poly A + poly U the maximum rate occurs 40°C below T m- the melting temperature; for DNA renaturation, 25°C below T m; and for RNA + DNA, 15 to 20°C below T m (Figs l(b) and 14). The apparent second-order rate constants are: (a) for poly A + poly U, 1·5 X 106 l.fmole sec at 34°C in 0·5 M-NaCI; (b) for renaturation of Diplococcus pneumoniae DNA, 21.jmole sec at 67°C in 0·3 M.NaCI, 0·03 M-sodium citrate; (c) for T2 RNA + T2 DNA, 10 l.jmole sec at 67°0 in 0·5 M-KCI-Q·Ol M-tris, pH 7·3 (if 50% of the T2 DNA is considered incapable of binding RNA, this becomes 20 l.jmole sec). In all three cases the units of concentration used are molarity of nucleotides and not the molar concentration of nucleic acid molecules. It is evident that, under equivalent conditions, DNA renaturation and RNA-DNA complex formation occur at similar rates, but that both reactions are several orders of magnitude slower than the reaction of poly A with poly U. The slowness of the RNA-DNA and DNA-DNA reactions appears to be a consequence of the requirement for bringing together complementary sequences. This would cause the reactions to be slower than that of the simple homopolynucleotides for at least two reasons. (I) They would be slower because the rate of reaction of any given sequence is governed not by the over-all DNA concentration, but by the much lower concentration of the one complementary sequence. If this were the only reason for the slow apparent rate, one might conclude that the T2 RNA-T2 DNA reaction consists of nearly 105 separate and independent reactions. (2) In addition, the RNA-DNA and DNA-DNA reactions would be slower because of the many accidental sequence pairings which possess some homology and therefore can persist long enough to impede the exploration needed to arrive at the "correct" base-pairing. The dependence on temperature of the rate at sub-optimal temperatures may be related to accidental pairing. The activation energy calculated for the region of maximum positive slope is: 3·5 kcal.jmole for poly A + poly U (in 0·5 M-NaOI), 20 koal.jmole for pneumococcal DNA renaturation (in 0·3 M·NaOI-Q·03 M-sodium citrate), and 23 kcal.jmole for the RNA-DNA reaction (in 0·5 M-KCI-Q·Ol M-tris, pH 7,3). Thus the two reactions which involve matching of complex nucleotide sequences require more energetic conditions, apparently in order to overcome some barrier to reaction. This barrier may be the restriction of conformational freedom caused by accidental pairing of the polynucleotide chains. We gratefully acknowledge the rapidity and precision of Kathleen Kliewer in helping us to obtain rapid kinetic data. This investigation was supported by a PHS research grant (A 3086) from the National Institute of Arthritis and Metabolic Diseases, Public Health Service. One of us (A. P. N.) would like to express his gratitude to Dr. H. O. Halvorsen for making possible his tenure in the Department of Microbiology of the University of Illinois. REFERENCES Bautz, E. K. F. & Hall, B. D. (1962). Proc, Nat. Acad. Sci., Wash. 48, 400. Geiduschek, E. P., Nakamoto, T. & Weiss, S. B. (1961). Proc, Nat. Acad. Sci., Wash. 47, 1405.
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Hall, B. D. & Spiegelman, S. (1961). Proc, Nat. Acad. Sci., Wash. 47, 137. Ha.ll, B. D., Green, M. H., Nygaard, A. P. & Boezi, J. (1963). 0014 Spr. Harb . Symp. Quant. B iol. 28, 201. . Marmur, J. & Doty, P. (1961). J. Mol. Biol. 3, 585. Nygaard, A. P. & Hall, B. D. (1963). Biochem, Biophys. Res. Oomm. 12, 98. Ross, P. D. & Sturtevant, J. M. (1962) . J. Amer. ahem. Soc. 84, 4503. Volkin, E. & Astrachan, L. (1956), Virology, 2, 149. Wa.tson, J . D. & Crick, F. H. C. (1953). Nature, 171, 737. Yankofsky, S. A. & Spiegelman, S. (1962). Proc. Nat. Acad. WaBh.48, 1069.
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