Stabilization of the native tertiary structure of yeast tRNALeu3 by cationic metal complexes

J. Mol. Biol. (1975) 97, 519-532

Stabilization of the Native Tertiary Structure of Yeast tRNA~" by Cationic Metal Complexes R. L. KARPEL, N. S. ]~LLER, A. M. LESK~ AND J. R. l ~ E s c o

Delaartment of Biochemical Sciences Frick Chemical Laboratory Princeton University Princeton, N.J. 08510, U.S.A. (Received 10 March 1975, and in revised form 12 June 1975) Complexes of inert (slowly exchanging) multivalent transition metal cations and neutral ligands were found to effectively replace Mg 2+ as stabilizers of the biologically active "native" form of yeast tRNA~ *u. From an analysis of the sigmoidal dependence of the denatured ~ native conformational equilibrium on complex cation concentration at 25°C in 0.15 M.KC1 at p H 7-4, it is calculated that about five complex cations must be tightly bound to stabilize the native conformer. Since this number is larger than the three binding sites previously observed for alkaline earth metal ions and polyamines, there must be sites of high negative charge on the tRNA accessible to Mg 2+ and spermidine s+ but not to the more bulky complex cations. In that case, the complex cations must occupy additional sites in order to stabilize the native conformer. The relative effectiveness with which the cations stabilize the native conformation is: Co(NH3)83+ >~ Pt(en)2(en-H) s+ > Co(en)33+ > Ir(NH3)sCI2+ --_~ Pt(NH3)~ 2÷. This order is consistent with general electrostatic considerations. Hydrogen bonding of the co-ordinated amines to tRNA phosphates could enhance the vaTmity of Co(NH3)ez+ • Since the complex eation-tRNA association constants are even larger than those of the cobalt complexes with multivalent inorganic anions, a cluster of at least three phosphates could constitute the bind. ing site of each tightly bound cation in tRNA. 1. I n t r o d u c t i o n Among biological macromolecules, nucleic acids are distinctive in possessing a unit negative charge a t every residue. Because of this high charge density, the stability of double helical secondary structure is markedly dependent on the presence of charge neutralizing cations (Dot:? et al., 1959). Moreover, since some negative phosphates m u s t be brought into unusual proximity when double helical segments of R N A fold to give rise to a tertiary structure, t h a t folding is associated with a stringent requirement for cations (Fresco eta/., 1966). As shown, for example, b y Fresco et al. (manuscript in preparation), such a requirement exists for t R N A in its native (biologically active) conformation, and can be m e t b y a variety of cations when present in sufficient concentration. These include monovalent alkali metals, divalent alkaline earth and transition metals, and a series of di- and trivalent polyamiues Present address: Department of Chemistry, Fairleigh Dickinson University, Teaueek, N.J. 07666, U.S.A. 519

520

R. L.

KARPEL ET AL.

(Fresco et al., 1966; Higuehi & Fresco, manuscript in preparation; Ishida & Sueoka, 1968). In particular, a combination of Na ÷ or K ÷ (0.15 •) and Mg 2÷ or spermidine (0.01 M) at physiological concentrations and temperature serves this purpose. While many cations can theoretically bind to tRI~A, the binding is co-operative and especially strong at only a few sites on the macromolsenle (see also Cohn et al., 1969; Williek & Kay, 1971; Schreier & Sch~mmel, 1974; Danehin, 1972). These "strong sites" presumably correspond to the areas of high charge density that result from the close approach of phosphates of distal backbone segments. The precise nature of these strong binding sites is not well understood at present. Nevertheless, it is not unreasonable to p~opose a qualitative difference between binding of multivalent cations that stabilizes tertiary structure, and binding that stabilizes secondary structure. The regular helical array of phosphate groups in secondary structural elements is interrupted by tertiary structural chain folding, and the unique charge arrangements that result discriminate markedly among cations of different size and shape. Hence, cation-filled cavities of different geometries will occur. Such specific site binding of a few cations is not expected for the stabilization of secondary structure. To shed some light on the nature of the strong cation binding sites on tRNA, we have studied the binding of multivalent complexes of inert (slowly exchanging) transition metal ions. These constitute a class of cations of known size, shape and charge distribution, can be prepared as salts of simple anions, and may be useful in the preparation of heavy-atom derivatives of tRNA required for crystallographic work. In such complexes, the central, highly charged ( ~ 2 + ) metal ion is surrounded by tightly bound neutral ligands, e.g. ammonia or ethylenediamine. The i~ner coordination sphere can be totally occupied by these ligands, such that substitution by solvent or nucleic acid phosphate oxygen donors does not occur under normal experimental conditions. Thus, binding of these cationic metal complexes to nucleic acids must be outer sphere in nature, i.e. the neutral ligands prevent phosphate groups from co-ordinating directly to the central metal ion.~ In contrast to such behavior, alkaline earth metal ions can bind directly to the phosphates of nucleic acids so that the metal-oxygen distance is approximately the sum of the ionic radii of metal ion and phosphate oxygen. Nevertheless, particular negatively charged cavities containing several phosphate groups could bind alkaline earth ions by both inner and outer sphere interactions, with the latter type possibly involving water molecules in the inner co-ordination shell hydrogen-bonded to phosphate oxygens. In view of these considerations, the effectiveness with which several octahedral and square planar transition metal complexes stabilize the native (N) conformer of yeast tRNAaLeu (Lindahl et a~., 1966; Kowalski et al., 1971) was examined. This tRNA was selected because it is one of a class of such molecules whose conversion from a biologically inactive or denatured (D) structure to a native one on incubation in a suitable ionic medium is particularly slow (Fresco et al., 1966; Lindahl eta/., manuscript in preparation). Hence, tRNA equilibrated in a particular ionic medium could be assayed by enzymatic aminoacylation. While the assay medium contains 0.01 M-Mg2÷, significant renaturation does not occur during the brief assay incubation (Lindahl e~al., 1967). Using this assay, the relation between the extent of renaturation t Many non-complexed metal ions of high charge could not be nsed for such a study because they catalyze phosphodiester bond scission (Eiohhorn & Bu~zow, 1965; Butzow & Eichhorn, 1965,1971).

t R N A I N T E R A C T I O N W I T H METAL COMPLEXES

521

of t R N A and the concentration of free cation was used to obtain information on the number of tightly bound ions and the equilibrium constant of this interaction.

2. Materials and Methods Ca) Gomp/ez eat/o~ Hexammlnecobalt (III)chloride, [Co(N~s)e]C]3; aCluopentarnm~ecobalt (HI)nitrate, [Co(NH3)sH20](NO3)8; and chloropentamrninecobalt (III) chloride, [Co(NHs)sC1]CI~, were gifts from Professor T. G. Spiro. Tetrammlueplatinum (II) chloride, [Pt(NHs)4]C12, was a gift from Matthey Bishop, Inc. Tris(ethylenediamlne) cobalt (III) chloride, [Co(en)3]C13, was prepared according to Work (1946); Tris(o-phenanthroline)cobalt (III) chloride, [Co(phen)3]Cls, according to Pfeiffer & Werdelmann (1950) ; bis(ethylenediamine)gold (III) chloride, [Au(en)2]C13, according to Block & Baflar (1951); trls(ethylenediamine)platlnum (IV) chloride, [Pt(en)3]C14, according to Giedt & Nyman (1966) and then exhaustively recrystallized from HCI:H20; and chloropentammlueiridium (III) chloride, [Ir(NHs)6C1] C12, according to Schmidt (1970). I n these syntheses, starting materials were of reagent grade or comparable purity, and all other chemicals were reagent grade. Aqueous stock solutions of these complex cation salts were made based on weight measurements and confirmed when possible by conductivity measurements (Grinberg, 1962a). (b) t R N A Bakers' yeast tRNA was enriched in tRNA3Le~ and depleted of tRNA~ uu and tRNA~* by fractionation on Sephadex G100, first with tRNA~ e~ selectively denatured, then with tRNA~*" native. Homogeneous tRNA~ °~ was also used in some experhnents (Kowalski et oL, manuscript in preparation). Each tRNA in 0.15 ~-KCI, 0.01 ~-cacodylate, 5 × 10-5 ~. EDTA, p H 7.4 (D-solvent) was concentrated by passage through a short colnm~ of DEAE-cellulose, precipitated with cold ethanol, recovered by centrifugation, and washed several times with cold 85% ethanol to remove salts. After resolution in the same solvent, it was denatured by passage through a column of Chelex 100 (Bio-Rad) which removes any simple multivalent metal cations. (c) Reno~uro~m st~ie~ tRNA was dissolved in D.solvent also containing the desired concentration of the complex cation under study and, except where otherwise noted, was allowed to reach equilibrium at 25±0-5°C over 5 to 6 days. The fraction of N-tRNA~ e" at equilibrium was determined by the enzymatic ammoacylation assay (see below). The time for equilibrium attainment was selected after study had been made of the kinetics of renaturation for those complex cations that were examined quantitatively. To achieve maximum accuracy, each data point (cation concentration in equilibrium experiments or elapsed time in kinetic experiments) represents the average of duplicate samples from a separate sample, incubated in a sealed 3 mm (internal diameter) × 40 mr, Pyrex tube. (d) E n z y m o ~ assay for ~Zive tRNA~*" The assay was a modification of that previously described (Lindahl e~ o~., 1966). The assay mixture contained in 0"1 ml, 0.16 mg crude enzyme, 0.01 ~nol [l~C]leucine (0.3 to 1.1 ~Ci), 0.06 to 0.2 A2Bs units tRNA (i.e. 1 × 10 -e to 3.6× 10 -6 M), 5.5 ~mol cacodylate (K+), pH 7.4, 1.6 ~mol MgC12, 1"0 ~mol ATP, 9.5 ~mol KCI, 0"002 ~mol EDTA(K2). Incubation proceeded for 20 min at 25°C, except for experiments monitoring kinetics of renaturation, where it was 20 rain at 20°C. The procedure for aminoacyl.tRNA isolation and radioassay was as described by Lindahl e~oJ. (1966).

(e) Treatmen~ of data To estimate the fraction of tRNA~ eu renatured, specific activities corresponding to fully denatured and fully native samples were required. The activity of the former was obtained from samples equilibrated in the D-solvent for 6 days a t 25°C. Such denatured

522

R.L.

KARPEL

ET .4L.

samples showed activity levels just slightly above control assays without tRNA. That for native samples was obtained by equilibration in D-solvent also containing 0.01 m-MgC12 either at 25°C for 6 days or at 60°C for 5 rain, followed b y cooling to 0°C, both methods giving the same specific actim'tles. The level of renaturation achieved with any particular cation concentration was calculated by interpolation of the observed activity between the N and D levels. To assure that the metal complexes do not interfere with the assay, tRlqA samples renatured in the presence of both 0.01 M-Mg2 + and representative concentrations of the complex ions were assayed. Except where noted, the level of accepter activity was the same as that with the complex ion absent.

3. R e s u l t s

(a) ~ a l / t a 2 i v , rer~**ratio~ a u d i ~ t R N A ~ " was equilibrated b y exposure to the complex cations a t 60°C for 5 rain, conditions t h a t lead to complete renaturation in 0.01 M-Mg2+ (Lindahl et al., 1966). All the cations tested (see Materials and Methods, section (a)) except Co(NHs)sC12+, which was ineffective even at 2 × 10 -8 M and precipitated t R N A at higher concentrations, were found to renature to a signi~cant extent. A selection of the renaturing cations was then made for further s t u d y at 25°C: Co(NH3)68+, Co(en)s 8+, Pt(en)2(en-H) s+, Pt(NHs)42+, Ir(NH3)sC12+, An(on)23+. (b) Kine~/0 ,tudi,8 To assess the time required to attain conformational equilibrium a t 25°C, the kinetics of renaturation were exar, lned a t concentrations of these complex ions estimated to achieve at least 50~o N - t R N A . As found for simple cations, the kinetics of t R N A ~ u renaturation with the complex cations are first-order. A typical semilogarithmic plot is shown in Figure 1, and half-time values obtained with the different cations are given in Table 1. Though some variation in rate with different cations is evident, it is clear t h a t equilibrium is achieved at 25°C with all the cations tested within five days (the time allowed for the equilibrium studies).

FO0

0.I0

I

I0

,, I

2O

I •

3O

40

Time ( h )

FIG. 1. Semilogarithmic plot of renaturation kinetics of tRNAaLe" (8 × 10-e •) in the presence of 2.8 X 10 -8 M-(Ir(NH3)6CI)C12at 25°C. A o = initial amino acid accepter aotivity, A~ -----activitya~ time t, Ace = final (equilibrium) activity.

t R N A I N T E R A C T I O N W I T H METAL C O M P L E X E S

523

TABLE 1

Kir~etica of rencrt=ratior~ of ye~t tRN~4~ u o~ 25.0±0.1°G Cation

Concentration (~t)

Co(NHs)63 +

ti/= (h)

Co(on)3 ~+

4"92 X 1"75 ×

Pt(en)=(en-H) s +

4-51 × I0- 8~

5

I r ( l q ~ s ) . C 1 =+

2"8 X 10 -s 1"86 × 10 - =

8-5 4"5

Pt(Iq-H3)4=+

5"5 13

10- ~ 10-"

The solvent also contained 0.15 z~-KCI, 10 -a M-eacodylate, 5× 10 -6 ~r-EDTA (pH 7"4). Concentration of Pt(en)3C14 (see text). (c) E~ilibrium 8tz~iea A typical plot of the dependence of tRNA~ eu conformation on free complex cation concentration is shown in Figure 2. The simple sigmoidal nature of this dependence is reminiscent of t h a t observed with simple cations (Fresco et al., manuscript in preparation). Hence, the analysis used is based on the assumption t h a t the D form is converted to the N form on co-operative binding of n moles of cation. This leads to the expression log ~ - j = n log [C]~,oe + log KN/.,

IN]

KN/D =

where

~D] [C]~o

With IN] and [D] experimentally determined, the ~ ions strongly bound on renaturation were obtained from the slopes of plots of log [N][[I)] versus log [C]rreo. While I'00

/

0'80

/

Z

=_ o.60 0> ==

(>20

/ /

I 0.002

I

I 0"004

!

[(,~c.H~.cOc,.]c.~,o-'~

0'006

FIG. 2. Dependenoe of [N]/[D] equilibrium of t R N A ~ eu on (Ir(NH3)sCI)CI 2 concentration at 25°C. The points are experimental and the solid line is ealoulated {see text).

524

R. L. KARPEL

~2'

.4L.

[O]~roe was not determined, the concentration of complex cation was set to be in sufficient excess of tRNA concentration so t h a t [C]~roe ---- [C]to~l. This was confirmed b y showing (a) t h a t the level of renaturation was invarJant over a twofold concentration range of enriched tRNAsLeu and (b) t h a t the concentration of cation giving 50~/o renaturation was the same when homogeneous tRNAsLeu was used at a 10 to 50-fold lower t R N A concentration. This latter behavior was displayed b y all cations except Pt(NHs), =+ (see below). Criterion Ca) did not hold in the case of Co(NHs)6 s+, and for the lower range of Pt(en)2(en-H) 3+ data (these ions renature at particularly low concentrations). I n fact, even at the lowest [Co(NH3)es+Stotal:KtRNA] testable, a significant portion of the cation is bound b y tRNA, and renaturation occurs. For example, with [tRNA] = 8.0× 10 -e M and [Co(NH~)6s+] = 3.0× 10 -5 M, giving the low molar ratio of 3.7, 23% renaturation occurs. Although for this reason n cannot be accurately determined, it is reasonable to assume t h a t it is similar to the values found for the other octahedral cations, about five. Hence, in the above example, the free cation concentration must be exceedingly low. The linear dependencies of log [N]/[I)] on log [Cirri= required b y the assumed model are generally met at least to 5 0 ~ N b y all the complex cations studied, as seen in Figure 3 (though the plots for Co(NHs)6 s+ and possibly Pt(en)2(en-H) s+ are subject to the ]imitation noted). A least-squares minimization computer program was used to obtain the lines for the octahedral cations. I t was observed t h a t when those data points that deviate most are excluded from the calculation, the correlation eoemcient increases significantly, to 99~/o, except for Co(en)3 s+, for which it is increased to 97 ~/o. However, these exclusions did not change the slopes b y more than ±0.5. Data points for cation concentrations beyond the range of the co-operative transition also were not included. I t is evident from Figure 3 t h a t the ]eve] of renaturation falls short of 1 0 0 ~ for several of the cations and decreases thereafter. This behavior is likely indicative of the formation of t R N A aggregates induced b y the complex cations. 25"0(

!o.95

IO'(X

! le I

/,

5`0C

4"

t

'I.OC 0.5C

0.10 0.07 I0 "s

=

.

A

f-

~'90

I I

-3.75 ~

;,.

I|

-

~'50 c .o

fli I =l,J= .10 -4

=

i

= I ==J

- 0"25

- .0'I0 ,

I

I

iO-a

[Complex cation] (M)

FIG. 3. Dependence of log [N]/[D] equilibrium of tRNA~ u on log oomplex cation ooncencration af~ equilibrium at 25°0. - - 0 - - 0 - - Co(IN-H3)68+; - - V - - V - - Pt(en)=(en-H)~+ m A - - A ~

0°(en)s 3+ ; - - ( D - - ( D - - ]Cr(N'Hs)sC]-=+; ( l ) , Pt,(NHs),=+.

t R N A I N T E R A C T I O N W I T H METAL C O M P L E X E S

525

Values for n, log K~/D, and [C]N=D, the cation concentration needed to attain 50~/o renaturation of t R N A ~ u a t 25°C, are given in Table 2. Note t h a t the slopes cluster around 5 for the octahedral cations. I n the case of the square planar, Pt(NH3)42+, a reliable value of n could not be obtained. The level of renaturation effected b y this cation varied with the t R N A sample. The d a t a shown in Figure 3 were obtained with the enriched sample of tRNA~ eu. W h e n homogeneous t R N A ~ u was employed, the d a t a shown in Figure 3 were reproduced for the other cations. With Pt(NHs)42+, some renaturation did occur at the lowest cation concentration studied, 7-3 × 10- 4 M. However, the native level only decreased as [Pt(NH3)42+] was raised to a point still less t h a n the lowest shown in Figure 3. Recognizing t h a t this behavior might be due to a low level of chemical reactivity, via substitution of the - N H 3 ligands b y residues on the t R N A , or b y oxidation of the central P t ( I I ) ion to a higher oxidation state, dithiothreitol (10 -3 M) was added in one experiment to the pure tRNA~ ~u before attempting to renature. This resulted in a level of renaturation like t h a t shown in Figure 3 for the enriched t R N A ~ u,

TABLE 2

Renaturation of tRiVA~ e" by complex orations at 25°C$ Cation

Co(NH3)~s + Pt(en)2(en-H) 3+ Co(en)38 + Ir(NH3)6C12+ Pt(NHs)~2+ Mg2+ ~ Ca2+~ Spermidine 8+~

Concentration at 50% renaturation (M) < 3.7 × i0- 5 <3.9 × 10-5 1.68 × 10-4 1.62 × 10 -8 ~ 2 × 10-3§ 2.9 × I 0 - 4

1.8 x 10 -4 9.5 × 10 -4

log K

23-6 23.3 17.8 12.8 ~9§ 11-3 13-1 9.1

5-3 5.3 4-7 4.6 3-5§ 3-2 3-5 3

t The solvent also contained 0-15 •-KC1, 10 -2 ~-eacodylate, 5 × 10 -6 M-EDTA (pH 7.4). Data from Fresco e~ al. (manuscript in preparation). § See text. suggesting t h a t one of the foregoing explanations did apply. While the d a t a with Pt(NHs)42+ are therefore not appropriate for quantitative comparison, it is evident t h a t this cation can serve as a n effective renaturant, p r o b a b l y a t a similar level to Ir(NH3) sC12+. The d a t a for [C]N=D indicate t h a t the relative effectiveness for stabilizing N - t R N A ~ u is Co(NH3)68+ ~ Pt(en)2(en-H) 8+ > Co(en)88+ > Ir(NH3)sC12+ - Pt(1VH3)a2+, with effectiveness ranging over two orders of magnitude. N o t e t h a t three of these cations are substantially more effective t h a n the likely biological stabilizers, Mg ~+ and spermidine 3+. I n keeping with the negative enthalpy change associated with the D - > N transition (Lindahl eta/., manuscript in preparation), it was observed in preliminary experiments t h a t higher cation concentrations are required for renaturation a t elevated temperatures.

526

R.L.

KARPEL

ET AL.

(d) 10H v a r ~ of i~(~)sG'~a.ind~ee~ r , ~ r e ~ i o ~ Pt(en)sC1~ exists in aqueous solution in several protolytic species with pKa values of 5.5, 9.7 and 10.7 (Grinberg, 19625). Below p H 5-5, the tetrapositive Pt(en)s 4÷ form predominates; above this pH, the Pt(IV)-bound ethylonediamlne nitrogen ligands sequentially deprotonate. Thus, during renaturation at pH 7.4, the dominant free species is Pt(en)2(en-H) 8~, whore on-H is H~NCH2NH2NH-. However, this does not necessarily mean that the tripositive species is the renaturing cation. To clarify this question, the pH dependence of renaturation with Pt(en),C14 at 25°C was examined under otherwise standard conditions. However, the pH range of these experiments was necessarily limited because the metastability of the D-conformer of tRNAaLeu is lost below pH 5 (Fresco et aL, 1966; Lindahl et aL, manuscript in preparation). After preineubation at p H 5.4, 6-4 and 6.7, samples were brought ¢o p H 7.4 and immediately assayed. (Control tRNA samples preincubated at these pH values in 0.01 ~-Mg ~+ all showed 100~/o levels of renaturation; of course, this concentration is well in excess of that needed for the D-->N conversion, and may therefore be insensitive to any pH dependence of the conformational equih'brium.) At a Pt(en)aC1 ~ concentration of 2.9× 10 - 6 M, the level of renaturation at both pH 6.7 and 7.4 was 3 ~ . At 3.5 × 10 -8 m, the level at pH 6.7 (12~) was even lower than at pH 7.4 (25~); and this trend was maintained when the concentration was raised to 4.2×10 -5 m, which yielded 58%, 8 1 ~ and 79~/o renaturation at pH 5.4, 6-4 and 7.4, respectively. If the binding of Pt(en)a t + to tRNA is signi~cantly stronger than that of Pt(en)2(en-H) 8÷, one would expect higher levels of renaturation in more acidic samples, and this is clearly not the case. Thus Pt(en)2(en-H) 3÷ must be the major binding species at pH 7.4. The slight drop in renaturation at lower pH values probably indicates that the tRNA eonformational equilibrium is somewhat sensitive to protona~ion of base residues.

(e) tRNA-Au(en)~ + interaction Au(on)~C18 has been effective in the heavy-atom derivatization of yeast tRNA co-crystals (Wright et aL, 1972), although in solution its interaction with tRNAa~eu appears to be complex. Prolonged incubation of Au(en)~8+ with tRNA at 25°C gave no evidence of renaturation, whereas brief incubation at 60°C showed considerable variation in the extent of renaturation at given cation levels between 2)< 10 -5 M and 1 )< 10-4 M. No renaturation was observed below 2)< 10-5 M, and above 1 × 10 -4 M the Au(en)28÷ was seen to interfere with the aminoacylation assay. Since Au(HI) complexes are generally less inert to ligand substitution than the other cations studied (Basolo & Pearson, 1967; Wilkins & Popplewell, 1956), covalent A u - - N bonds could form by reaction of Au(en)23÷ with tRNA nucleotides or synthetase basic amino acids (Gibson e~ a~., 1971). The non-reproducibility of Au(en)23÷dependent renaturation might also be related to the strong oxidizing action of Au(III) compounds (Cotton & Will,in,on, 1966); faint yellow stock solutions of Au(en)~C13 were seen to develop deep purple precipitates on storage, even in the dark and at <0°C. 4. Discussion The stabilization by complex metal cations of the native conformer of yeast tRNAaI~u parallels the behavior of alkaline earth metal ions and polyamines (Fresco et al., manuscript in preparation). Thus, there is a sharp single transition between

~RNA

INTERACTION

WITH

METAL

COMPLEXES

527

the inactive conformer and the native conformer over a narrow range of cation concentration, that is indicative of co-operative binding of a small number of ions. Determln~tion of this number, n, is made accessible by using the amlnoacylation probe.

(a) N~mber of bindi~j s~cs The observed ~ ~ 5 for the hexa-co~ordinated complex cations is larger than the value of ~ 3 for 1 ~ 2÷, Ca 2÷, and the polyamlne, spermidine. Although the uncertainty of ±0.5 somewhat reduces the significance of this difference, the uniformly higher slopes of the oetahedral cations would seem to argue that the disparity is real. Moreover, the disparity is not correlated with amnlty per site on tRNA (see Table 3, and below). Since it seems reasonable that all these cations stabilize the same N conformer, how then this variation in ~? is formally the increment over those cations that may already be bound to the denatured conformer. Differences between observed ~ values could be due to a variation in binding to the D form, with a constant number of ions bound to the N form. However, it seems unlikely that the D form has sites at which cations are strongly attached. The D form has been found b y hydrodynamic methods to be a relatively expanded structure, suggesting that distal helical segments do not interact to the extent they do in the N conformer (Fresco ~ aJ., 1966). I t therefore seems ~m]i~ely that the regions of high charge density present in the N form exist in the D species. Moreover, the amn~ty of these cations for phosphates in double helices is very low in the ~ ~ 0.15 M medium in which the experiments are performed (Karpel e~ a~., manuscript in preparation), l~inally, we note again that the D -~ N conformational change is effected by two cations, Co(NH3)e8÷ and Pt(en)2(en-H) 8÷, at extremely low levels. Below the threshold of the transition the concentration of these complexes is comparable to that of the (denatured) tRNA. Were cations to bind to the D form, the a~nlty constants would necessarily be extremely high. Hence, while the technlques employed in this study do not directly rule out the possibility of some strong cation binding to the D form, we feel that this is un|ikely. Therefore, we regard ~, the number of tight binding sites, as an absolute rather than an incremental value. The variation in ~ can more reasonably be explained by the following argument. Let us consider q anionic sites of especially high charge density on the native molecule, which consequently exhibit unusual amnlty for cations in general and multivalent ones in particular. When Mg2÷ is the multivalent cation present, it takes binding to about three such sites to lower the electrostatic free energy sufllciently to stabilize the N conformer. If the complex cations could bind to the same three sites, then would equal 3 for them as well. Since ~ ~ 5 for the octahedral complexes, some of which have even greater s.~nlty for tRNA than Mg2+, the binding to at least one of the Mg~÷ sites must be either blocked or extremely weak. Binding to others of the sites is therefore required. The size of hydrated 1 ~ 2÷ (Mg(H20)~~÷) is quite similar to that of the Co(III) and Pt(IV) complex cations (Phil|ips & Williams, 1966; Nakatsu, 1962; Bokii, 1951). In the case of Mg~÷ binding to tRNA, however, ionic interaction with tRNA phosphates can involve removal of some of the hydration shell to allow direct metal-phosphate co-ordination. This is not possible for the six co-ordinated complex cations, whose inner co-ordination spheres are fully occupied b y tightly bound, non-substitutable

528

R.L.

KARPEL

ET

AL.

neutral ligands, ammonia or ethylenediam~ne. Hence, these complex cations are effectively larger and may not fit into a~ the Mg2+ binding sit~s. (b) Relative b~nding a~nitie8 The relative effectiveness with which the cations stabilize the N conformer of tRNA~ eu (see Table 2) is consistent with general electrostatic considerations. The tripositive charged complexes bring about the conformational change at significantly lower concentrations than the divalent cations. Pt(IV) complexes are about the same size as analogous Co(III) cations (Bokii, 1951; Nakatsu, 1962). Therefore, the ability of Pt(en)sCl 4 to effect renaturation at one-quarter the concentration of Co(en)3Cl.~ suggests that the effective charge on the major Pt(IV) binding species, Pt(en)2(en-H) s+, is greater than that of Co(en)33+, even though the formal charge of both cations is the same. The negative charge of the (en-H) -z ligand is presumably localized, and the orientation of Pt(en)2(en-H) 3+ at a binding site will most likely be optimal when this ligaud is located at the greatest possible distance from the phosphate groups. The effective charge of the complex would then be increased, leading to enhanced binding a~n~ty. In all likelihood, Ir(NHs)sC12+ binds in a similar manner, with its C1- ligand oriented away from the phosphates. In addition to electrostatic considerations, other factors may affect the binding amnlties to tRNA. Conceivably, hydrogen bonds between amino hydrogens and phosphate oxygens add stability to these interactions (Mason & Norman, 1964). Water molecules hydrogen-bonded between these groups could also increase the amnity. The ability of the am~no groups of Co(NH3)63+ to freely rotate, and thereby easily accommodate to the unique arrangement(s) of phosphates at the binding sites, might explain the greater affinity of this complex for tRNA~ e" than that of Co(en)38+. For the latter cation, the chelation of the ethylenediamine ligand prevents such free rotation. It is also possible that differential hydrogen-bonding abilities might cause one enantiomer of Co(en)38+ to bind more tightly than the other (in the present work, racemic mixtures were used). Such specificity, however, was not observed in a binding study of (~-) and (--) Co(en)38+ with DNA (Ascoli eta/., 1972). Differential hydrogen-bonding abilities between the asymmetrical Pt(en)~(en-H) 8+ and the symmetrical Co(en)33+ might also account for the observed differences between the binding affinities of these complexes. (c) Interpretation of binding a]finities Some insight into the nature of the binding sites on tRNA for cations is derived from the following considerations. The free energy associated with the D - > N transition can be partitioned into a free energy change on going from denatured to native conformer in the absence of cation binding, and the free energy change due to the binding of n cations to the native conformer, thus:

,,dG°N/D = .dG°~_c~/cD_C~ ~ Since

ziG°z~/D "< 0 ,dG°cN-c~IcD-c~ > 0t,

AG°cl~.

tRNA INTERACTION

WITH METAL COMPLEXES

529

then

,~G°c/N< 0

IAO°o I > lZe%_o, (o_o,1. Thus

Iz1(7%/~1 > ]ziG%/.[. T h a t is, t h e a b s o l u t e v a l u e o f t h e free e n e r g y c h a n g e o f c a t i o n b i n d i n g t o t h e n a t i v e c o n f o r m e r is g r e a t e r t h a n t h a t o f t h e o v e r a l l r e a c t i o n . T h e a v e r a g e free e n e r g y o f b i n d i n g p e r site o n N - t P ~ A a Leu is, therefore, o f g r e a t e r m a g n i t u d e t h a n a n a r b i t r a r y q u a n t i t y t h a t w e define as t h e o v e r a l l free e n e r g y c h a n g e p e r s i t e :

T h e n , t h e average site e q u i l i b r i u m c o n s t a n t (there m a y b e c o n s i d e r a b l e v a r i a t i o n a m o n g t h e sites) 1

Kslte ---- (KZ~/D)It" "~ exp(--AG°N/D/nRT) =

[C]~=D"

I t s h o u l d b e r e c o g n i z e d t h a t t h i s c o n s t a n t r e p r e s e n t s a lower l i m i t for t h e t r u e c a t i o n site b i n d i n g c o n s t a n t b e c a u s e

I a% l < Ina°

l.

V a l u e s for Kalto a r e g i v e n in T a b l e 3. TABLE 3

Cation-t R N A site equilibrium constants Cation Co(NHa)83 + Pt(en)2(en-H) 8+ Co(en) 8a+ Ir(17Hs)sC1 ~+ Mg~+t Ca~+t SPermidines + t

K~,t. × 10 -2 (~-1)

logKs,to

> 270 >_250 61 6-3 35 56 11

> 4-43 _>4-40 3-79 2.80 3-54 3-75 3.0

2 5 ° C , g -~ 0.15 M-KC1. Data from Fresco st a~. (manuscript in preparation).

t =

~ o r c o m p a r i s o n , we list in T a b l e 4 s o m e r e p r e s e n t a t i v e s t a b i l i t y c o n s t a n t s o f s i m p l e a n i o n s w i t h b o t h Co(NH3)s a÷ a n d Co(en)83÷. W h e r e a s log Kslte for t h e s e t w o c a t i o n s o b t a i n e d w i t h t R N A ~ u is a b o u t 4 (a lower limit), t h e v a l u e s s h o w n i n T a b l e 4 for unto a n d d i n e g a t i v e ions a r e g e n e r a l l y less t h a n 2 i n s o l v e n t s o f s i m i l a r ionic s t r e n g t h . T h o u g h log K v a l u e s are s o m e w h a t l a r g e r for t h e cyclic tri- a n d t e t r a m e t a p h o s p h a t e s a f t e r n o r m a l i z a t i o n to s o l v e n t s o f s i m i l a r ionic s t r e n g t h , t h e y a r e still t This inequality is relevant to tRNAs under a range of conditions where they are denatured. For some tRNAs, such as tRNA~ eu, this is so at 25°C in g = 0.15 in the absence of multivalent cations; but some tRNAs appear to be already native under these conditions. I n those eases, ~G°¢N_c)/(~_c) < 0, where I) represents non-native conformers, e.g. "extended" forms. For such ~RNAs, ~ = 0 formally, but q sites for tight binding of cations should still exist. 8o

R. L. K A R P E L

530

E T AL.

TABLZ 4

Some a~ni~y c~n~tanf8 of Oo(NHz)/ + and Oo(en)z3 + with 8ir~le anion~t Anion

Cation

log K~;

C1BrBr-

Co(NHs)e s + Co(NHs) ss + Co(en)ss +

SO, ~-

Co(NHs)s s +

SO42SO4s P309 s P30g 3P40124-

Co(NH3)e" + Co(en) ss + Co(NHs)s s+ Co(en)s~+

0"34 1"66 1-32 2"06 1.84 2.01 4"44 4.46§ 5"74

P4Ox~ 4 Fe(CN)s 4-

Co(N-Hs)s 8 + Co(en) s 3 + Co(on)a s+

5"77 § 2.0

Conditions: t,/~ 25°C, 25°C, 25°C 25°C 25"C 25°C 25°C 25°C 25°C 25"C 20°C

0-07 M-NaCtO~ 0-054 M-NaC104 0-3 M-NaCIO4 0.07 ~r-NaC104 0-1 M-NaC104 0-07 ~-NaC104 ~ -~ 0~ ~ -~ 0¶ ~ -~ 0¶ ~ -~ 0¶ 0.21 x-NaC104

Except where noted, data are taken from Sillen & Martell (1964,1971). [Ion -- Pair] K = [Complex cation][Anion] ' § Monk (1952). ¶ Extrapolated to zero ionic strength. Using the Fuoss equation (Fuoss, 1958; Hammes & Steinfeld, 1962) with an interionie distance of 6 A, we calculated that the log K values for P309 s and P~Ox24- at p = 0.15 are 2.37 and 3.25, respectively. At p = 0, this equation yields values of 4-38 and 5.92, very close to the experimental values. Note that these anions are cyclic.

s u b s t a n t i a l l y less t h a n t h e m a g n i t u d e o f t h o s e d e t e r m i n e d w i t h t R N A . Hence, t h e b i n d i n g t o t R N A is m u c h s t r o n g e r t h a n t o t h e tri- a n d t e t r a n e g a t i v e small anions. Since such s t r o n g b i n d i n g t o t h e D c o n f o r m e r does n o t occur, t h e t i g h t l y b o u n d c a t i o n s m u s t n o t i n t e r a c t s i m p l y w i t h p h o s p h a t e s o f t h e d o u b l e helical c l o v e r l e a f segments. R a t h e r , t h e y m u s t b i n d a t sites on t h e n a t i v e t R N A w h e r e s e v e r a l phosp h a t e s a r e e v e n m o r e closely clustered. I n a d d i t i o n t o Coulombic a t t r a c t i o n (which m u s t p l a y t h e m a j o r role i n s m a l l i o n i n t e r a c t i o n s ) a n d t h e p o s s i b i l i t y o f h y d r o g e n bonding, closely s p a c e d p h o s p h a t e s in a m a c r o m o l e c u l e w i t h s t a b l e t e r t i a r y s t r u c t u r e could i n t e r a c t w i t h c a t i o n s b y outer s p h e r e chelation, w i t h h i g h e r s t a b i l i t y r e s u l t i n g from the entropical]y favorable displacement of water from the tRNA and/or cation t. P h o s p h a t e clusters could occur w h e r e d i s t a l c l o v e r l e a f loops i n t e r a c t , a n d w h e r e a d j a c e n t single s t r a n d e d c h a i n s e g m e n t s fold t o f o r m t h e n a t i v e t e r t i a r y s t r u c t u r e . This w o u l d a c c o u n t for t h e s t r i n g e n t r e q u i r e m e n t for c a t i o n s t a b i l i z a t i o n o f n a t i v e t R N A . I n v i e w o f t h e g e n e r a l i t y o f s u c h ionic r e q u i r e m e n t s , t h e s e r e s u l t s w i t h t R N A ~ u m a y also b e v i e w e d as g e n e r a l (Fresco e~ al., 1966). H o w e v e r , t h e p a r t i c u l a r r e q u i r e m e n t s a r e l i k e l y t o v a r y a c c o r d i n g t o u n i q u e p r i m a r y s t r u c t u r a l features, s u c h as v a r i a t i o n s in t h e sizes o f t h e " e x t r a a r m " a n d t h e h U loop, a n d t h e p r e s e n c e o r absence o f p o s i t i v e l y c h a r g e d residues in t h e e x t r a a r m a n d i n t h e T~F loop, w h e r e mTG ÷ a n d m l A + a r e s o m e t i m e s p r e s e n t (Staehelin, 1971). T h e s e r e s u l t s a r e n o t We may consider a set of clustered phosphates on the tRNA as comprising a multidentate ligand (i.e. being able to form several bonds with the cation). When these phosphates, which are covalently fixed on the same molecule, bind to a complex cation, they must liberate ordered water molecules that were bound in the cation's outer sphere. This binding is analogous to the inner sphere formation of a chelate when a multidentate ligand (e.g. EDTA) attaches directly to a simple metal ion, resulting in the liberation of water molecules that had been fixed in the ion's (now occupied) ~;n~er sphere.

t R N A I N T E R A C T I O N W I T H METAL C O M P L E X E S

531

inconsistent with the localization of several h e a v y cation sites in crystals of yeast t R N A T M (Kim et al., 1972,1973; Robertus et al., 1974) or in co-crystals of yeast t R N A (Wright e~ al., 1972). We are grateful to Ms Rosina T. Wilson for technical assistance and to Dr T. Spire for helpful discussions. T h i s work was supported b y grants from the National Institutes of Health (GM-07654), the National Science Foundation (GB-35595), and the American Heart Association. One of us (R. L. K.) was a United States Public Health Service postdoctoral fellow (1971-72). REFERENCES Ascoli, F., Branca, M., Mancini, C. & Pispisa, B. (1972). J. Ghem. Soc. ~araday, I, 68, 1213-1226. Basolo, F. & Pearson, R. G. (1967). In M e c h a n ~ . s of In~rganio Reaction~, 2nd edit., pp. 410-414, Wiley, New York. Block, B. P. & Baflar, J. C., J r (1951). J. Amsr. Ghem. See. 73, 4722-4725. Bokii, G. B. (1951). Izv. Akad. Naul¢, U.S.S.R. 15, 170-175. Butzow, J. J. & Eichhorn, G. L. (1965). Biopolymers, 3, 95-107." Butzow, J. J. & Eiehhorn, G. L. (1971). Biochemi~ry, 1O, 2014-2019. Cohn, M., Danchin, A. & Grunberg-Manago, M. (1969). J. Mol. Biol. 39, 199-217. Cotton, F. A. & W~ll~n~on, G. W. (1966). I n Advanced Inorganio Ghemistry, 2nd edit., pp. 1049, Interscience, New York. Danchin, A. (1972). Biopolymers, U , 1317-1333. Dory, P., Boedtker, H., Fresco, J. R., Haselkorn, R. & L i t t , M. (1959). Prec. Nat. Acad. Sci., U.S.A. 45, 482-499. Eichhorn, G. L. & Butzow, J. J. (1965). Biopolymera, 3, 79-94. Fresco, J. R., Adams, A., Ascione, R., Henley, D. & Lindahl, T. (1966). Cold Spring Harbor Syrup. Quant. Biol. 31, 527-537. Fuoss, R. M. (1958). J. Ame.r. Chem. Soy. 80, 5059-5061. Gibson, D. W., Beer, M. & Barrnett, R. J. (1971). Biochemi~txy, 1O, 3669-3679. Giedt, D. C. & Nymph, C. G. (1966). Inorganio Syntheses, 8, 239-241. Grinberg, A. A. (1962a). In A ~ Introduction to the Chemistxy of Complex Compounds, p. 62, Addison-Wesley, Reading, Mass. Grinberg, A. A. (1962b). In A n Introduction to the Chemiet~1 of Complex Compounds, p. 243, Addison-Wesley, Reading, Mass. Hammes, G. G. & Steinfeld, J. I. (1962). J. Amer. Ghem.Soc. 84, 4639-4643. Ishida, T. & Sueoka, M. (1968). J . Biol. Ghem. 243, 5329-5336. Kim~ S. H., Quigley, G. J., Suddath, F. L., McPherson, A., Sneden, D., Kim, J. J., Weinzierl, J., Blattmann, P. & Rich, A. (1972). Prec. Nat. Acad. Sci., U.S.A. 69, 3746-3750. K~m~ S. H., Quigley, G. J., Suddath, F. L., McPherson, A., Sneden, D., Kim, J. J., Weinzierl, J. & Rich, A. (1973}. S~/ence, 179, 285-288. Kowalski, S., Yamane, T. & Fresco, J. R. (1971). Sc/enc6, 172, 385-387. Lindahl, T., Adams, A. & Fresco, J. R. (1966). Prec. Nat. Ar,ad. Sci., U.S.A. 55, 941-948. Lindabl, T., Adams, A., Geroch, M. & Fresco, J. R. (1967). Prec. Nat. Acad. Sci., U.S.A. 57, 178-185. Mason, S. F. & Norman, B. J. (1964). Prec. Ghem. See. 339-340. Monk, C. B. (1952). J. Ghem. Soo. 1317-1320. Nakatsu, K. (1962). Bull. Ghem. See., Japan, 35, 832-839. Pfeiffer, P. & Werdelm~nn, B. (1950). Z. Anorg. Ghem. 263, 31-38. Phillips, C. S. G. & W/lli~.m~, R. J. P. (1966). I n Inorganic Ghemistxy, part 2, p. 249, Interscience, New York. Robertus, J. D., Ladner, J. E., Finch, J. T., Rhodes, D., Brown, R. S., Clark, B. F. C. & Klug, A. (I974). Nature (London), 250, 546-551. Sehmldt, H. H. (1970). Inorganic S y n ~ e z , 12, 243-247. Schreier, A. A. & Scb~mrnel, P. R. (1974). J. MoL Biol. 86, 601-620.

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KARPEL

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Sillen, L. G. & MarteU, A. E. (1964). Eds of ,.~tabilit/y Constants of Metal-Ion Complexes and (1971) ,.~upp~ement No. 1, The Chemical Society, London. Staehelin, M. (1971). Expericntia, 27, 1-11. Wilkius, R. G. & Popplewcll, D. S. (1956). Rec. Tray. 75, 815-818. Wilhck, G. E. & Kay, C. M. (1971). Biochemistry, 10, 2216-2222. Work, J. B. (1946). Inorganic Synthesas, 2, 221-222. Wright, H. T., Karpel, R. L., Beurling, K., Blake, R. D., Subramanian, E., Fresco, J. R. & Langridge, R. (1972). Acta Crystallogr. A28, III-28.