J. Mol. Biol. (1978) 122, 321338

Nucleic Acid Binding Properties of Escherichia coli Ribosomal Protein Sl I. Structure and Interactions of Binding Site I DAVID E. DRAPER? AND PETER H. VON HIPPEL I&itute

of Molecular

Biology and Department University of Oregon Eugene, Oreg. 97403, U.8.A.

of Chemistry

(Received 20 k?eptember 197’7) coli ribosomal protein Sl plays a central role in initiation of protein synthesis, perhaps via participation in the binding of messenger RNA to the ribosome. Sl protein has two nucleic acid binding sites with very different properties: site I binds either single-stranded DNA or RNA, while site II binds acid binding single-stranded RNA only (Draper et al., 1977). The nucleic properties of these sites have been explored using the quenching of intrinsic protein fluorescence which results from binding of oligo- and polynucleotides, and are reported in this and the accompanying paper (Draper & von Hippel, 1978). Site I has been studied primarily using DNA oligomers and polymers, and has been found to have the following properties. (1) The intrinsic binding constant (R) of site I for poly(dA) and poly(dC) is ~3 x lo6 Mm1 at 0.12 ivr-Na+, and the site size (rz, the number of nucleotide residues covered per Sl bound) is 5-l & 1.0 residues. (2) Binding of site I to polynucleotides is non-co-operative. (3) The K value for binding of Sl to single-stranded polynucleotides is -lo3 larger than I< for binding to double-stranded polynucleotides, meaning that Sl (via site I) is a potential “melting” or “double-helix destabilizing” protein. (4) The dependence of log K on log [Na+] is linear, and analysis of the data according to Record et al. (1976) shows that two basic residues in site I form charge-charge interactions with two DNA phosphates. In addition, a major part of the binding free energy of site I with the nucleic acid chain appears to involve non-electrostatic interactions. (5) Oligonucleotides bound in site II somewhat weaken the binding affinity of site I. (6) Binding affinity is virtually independent of base and sugar composition of the nucleic acid ligand; in fact, the total absence of the base appears to have little effect on the binding, since the association constant for 2’.deoxyribose 5’-phosphate is a,pproximately the same as that for dAMP or dCMP. (7) Two molecules of d(ApA) can bind to site I, suggesting the presence of two “subsites” within site I. (8) Iodide quenching experiments with Sl-oligonucleotide complexes show differential exposure of tryptophans in and near the subsites of site I, depending upon whether neither, one, or both subsites are complexed u-ith an oligonucleotide.

Escherichia

1. Introduction Elucidation of the molecular details of the functioning of ribosomes during protein synthesis has been a challenging problem for molecular biologists for some years. The enormous complexity of the ribosome itself, together with the number of protein 7 Present address : Department of Molecular, of Colorado, Boulder, Col. 80302, U.S.A.

Cellular

and Developmental

Biology,

University

321 0022-2836/X4/1223-2138

$02.00/O

@J 1978 Academic

Press Inc. (London)

Ltd.

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and nucleic acid factors with which it interacts, have required various physical and chemical approaches to probe its assembly, a’rchitecture, conformational states, and interactions. In recent years, considerable insight into the details of protein-nucleic acid interactions have been gained from physico-chemical studies of simpler systems. For instance, studies of the interaction of lac repressor with double-stranded nonoperator DNA (e.g. see Butler et al.; 1977; Kao-Huang et al., 1977), and of phage T4coded gene 32 protein with single-stranded nucleic acids (e.g. see Jensen et al.; 1976 ; Kelly et al., 1976), have provided appreciable quantitative information which has helped to clarify some aspects of the functioning of these systems in viva. Since ribosomal assembly, structure, and function depend to a very large extent on the recognition and manipulation of ribosoma,l, transfer, and messenger RNAs: it seemed to us likely that some problems in ribosome function could also benefit from an application of the experimental approaches used in studies of simpler proteinnucleic acid interaction systems. Ribosomal protein Sl appeared to be a likely candidate for such a study. This protein is known to bind various RNAs independently of ribosomes (Tal et al., 1972), and numerous studies have indicated that Sl plays a crucial role in protein synthesis, possibly by facilitating the binding of mRNA to the ribosome (see, for example, van Dieijen et al., 1975;1976). A variety of hypotheses have been advanced to account for the effects of Xl on the first steps of protein synthesis. The first (and simplest) hypothesis is that an RNA binding site on Xl forms part of the messenger binding site on the ribosome (Tal et al., 1972). van Dieijen et al. (1976), argued from data on the binding of partially denatured mRNA to ribosomes that Xl aids mRNA binding by influencing the tertiary structure of the mRNA. Other hypotheses have centered on the proposal of Shine & Dalgarno (1974) that three to seven base-pairs: involving complementary pairing between the 3’ end of the 16 S rRNA and a region just preceding the AUG initiation codon on the mRNA, serve as a recognition mechanism in initiation. Dahlberg & Dahlberg (1975), noting evidence that Xl binds to the ribosome in the region of the 16 S rRNA 3’ terminus, and taking into account the possibility that secondary structures (hairpin loops) formed in this region of the rRNA could prevent the proposed base-pairing, suggested that the role of Sl is to melt out a hairpin in the 16 S rRNA, thus making the bases available for base-pairing pith the messenger. Steitz et al. (1977) suggested that Xl may be stabilizing this base-paired region once it is formed. Although Xl is known to remain on the ribosome during the entire protein synthesis process (van Duin & van Knippenberg, 1974), none of these proposals deals with the role of Xl in elongation. In addition Laughrea & Moore (1977) have recently shown that 30 S ribosomal subunits can, under certain conditions, bind two copies of Sl protein. The location and function of these multiple binding sites have not been studied. Thus the specific function(s) of Xl are largely unknown. A thorough understanding of the polynucleotide binding properties of Xl could and to lay the groundwork for further help to test some of these hypotheses, studies on the interactions of Sl with ribosomes and mRNA. In previous communications (Draper & von Hippel, 1976; Draper et al., 1977) we described the intrinsic fluorescence properties of Xl. and showed that this fluorescence is quenched by bound oligo- a,nd polynucleotides. Titrations with single-stranded ribo- and deoxyribopolynucleotides suggested that there are two classes of binding sites on Xl protein, and this conclusion was confirmed by titrations with nucleotide oligomers and binding

BINDING

SITE

I OF RIBOSOMAL

PROTEIN

Xl

323

studies using a sucrose-gradient band sedimentation technique. Site I was shown to bind either DNA or RNA oligomers and polymers, while site II was found to be highly specific for RNA binding; both sites are present on every copy of Sl protein. The presence of two binding sites opens the possibility that Sl could bind ribosomal and messenger RNAs simultaneously in vivo ; alternatively, it could cross-link two sections of the same RNA. Information on the binding mechanism and nucleotide specificity of each site is presented in this and the accompanying paper (Draper & von Hippel, 1978). The different specificities of the two binding sites make it possible to study each site separately. In this paper we examine the binding of DNA oligomers and polymers to site I using fluorescence titration techniques. In the accompanying paper we describe in detail the binding of RNA to site II, using a high concentration of DNA oligomers to saturate site I. The binding properties determined for the two sites are quite different, and suggest possible mechanisms of Sl interactions with ribosomes and mRNA.

2. Materials and Methods (a) Buffers Basic buffer refers to 20 mivr-Tris, 1 mM-NaaEDTA, 1 mM-2-mercaptoethanol, 10% (v/v) glycerol, and the indicated concentration of NaCl at pH 8.0; e.g. 0.1 M/BB represents basic buffer plus 0.1 M-NaCl. Standard buffer contains 10 mM-disodium phosphate, 1 rnx-Na,EDTA, 1 m&r-2.mercaptoethanol, 10% (v/v) glycerol, and NaCl as indicated (e.g. 0.1 M/standard buffer), at pH 7.7. All buffers were made up with water doubly distilled from glass. (b) 6’1 protein, S 1 protein was isolated from Escherichia coEi strain B (Grain Processing, Iowa) or strain MREBOO (Medical Research Establishment, England) as previously described (Draper et al., 1977). The purification procedure involves isolation of crude ribosomes and release of the Sl protein from the ribosomes by dialysis against low-salt buffer (Tal et al., 1972), followed by DNA-cellulose chromatography and sucrose gradient sedimentation in high salt solution. The yield of protein by this procedure is about 5 mg/50 g cell paste. The protein is free of nucleic acid (A,,,/A,,, = 1.65) and is better than 95% pure as shown by sodium dodecyl sulfate-gel electrophoresis. An extinction coefficient (at 280 nm) of 0.60 mg/ml per cm was used, based on Lowry protein assay (Lowry et al., 1951), and a molecular weight of 65,000 was calculated from the migration of Sl relative to protein standards in sodium dodecyl sulfate-gel electrophoresis. (c) Nucleotides from Calbiochem, and Mononucleotides (dAMP, dCMP, and TMP) were purchased 2’-deoxyribose B/-phosphate was obtained from Sigma. All oligonucleotides (d(ApA), d(pA),, d(pA),, and d(pA),) and polynucleotides (poly(dA) and poly(dC)) were purchased from Collaborative Research. Standard nomenclature is used for oligonucleotides : e.g. d(pA), refers to the deoxyribose oligomer pApA, where the leftmost phosphate is located on the 5’ hydroxyl. The oligoribonucleotide designated (rC)z was purchased from Collaborative Research, and refers to the average length of a mixture of oligonucleotides designated C(PC)~~-~~ by the manufacturer. Standard literature values were used for extinction coefficients: polymer values were obtained from the compilation of Janik (1971), and oligomer values from that of T’so (1974). (d) Fluorescence

titration

method

All titrations were performed in a Hitachi MPF-2A spectrofluorimeter using a 3 mm x 3 mm quartz cuvette. The following procedure was used for all titrations. Sl was diluted into the desired buffer to a final volume of 150 ~1 and a concn of 0.5 to I.0 pM. Mixing was 12

D. E. DRAPER

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accomplished by gentle inversion of the cuvette several times; vigorous stirring of the solution was avoided as it tends to promote aggregation of Sl protein. An identical cuvette containing approx. 1 PM-N-acetyl tryptophanamide in the same buffer was also prepared for use as a standard. The cuvettes were allowed to come to thermal equilibrium in the fluorimeter, which was thermostatically controlled at, 250°C. The fluorescence of the 2 samples was then alternately observed, using an excitation wavelength of 294 mm and an emission wavelength of 340 nm. To obtain titration data, 3-~1 portions of nucleotide were added in succession and mixed by several gentle inversions of the cuvette. Titrations in which nucleotide concentrations exceeded 50 mM, and thus absorbance of the excitation beam by the nucleotide became significant, were corrected for this “inner filter” effect (and dilution) by adding identical portions of nucleotides to the reference N-acetyl tryptophanamide solution. (e) Calculation

of binding

and quenching parameters

from

JEuorescence

data

The fluorescence, F, for each titration point was expressed in arbitrary units as the ratio of the Xl fluorescence to the N-acetyl tryptophanamide standard fluorescence (the latter corrected for dilution if titration portions had not been added to the sta,ndard). lJ’ was converted to fractional intrinsic fluorescence quenching, Q, by comparison with the initial fluorescence of the sample (PO): Q = 1 With plots

nucleotides or oligomers, the titration (Klotz & Hunston, 1971): Q-l

(F/F"). points

= (Q,,,‘“KL)-l

were

plotted

as “double

reciprocal”

+ Q&,

of free ligand, and Q,,, the quenching where L is the concentration at ligand saturation. K (the association constant) and Q,,, can be estimated from the slope and intercept of data plotted as l/Q versus l/L. If the binding is weak, L will be approximately equal to the total ligand concentration ; for other cases L was calculated taking into account the amount of bound ligand. This was done using an iterative technique: an initial Qmax value was estimated from a plot of and this value was used to calculate 1/L for each point. The revised l/Q versus l/Ltotal, data were then used for a new calculation of Q,,,. The process was repeated until the 10” X-I, at our change in Qmax and K became very small. For large values of K (>4x usual concentration of protein), Q,,, could be estimated directly from the plateau of the binding curve, and used to recast the data according to the formulation of Scatchard (1949). For calculation of binding constants from polymer titrations, the data were plotted as Scatchard plots and fitted to the (non-co-operative) “overlap” binding equation of McGhee & von Hippel (1974). For a ligand (protein) that covers more than one nucleotide residue on the polynucleotide lattice, the concentration of free nucleic acid binding sites will not be a linear function of bound protein, since one protein can cover more than one potential binding site. Thus both the site size (n) and the binding constant (K)must be taken into account explicitly in calculating a binding isotherm. Optimal values of K and n were obtained using calculated overlap binding isotherms and a standard least-squares iterative procedure.

3. Results (a) Polynucleotide

binding properties

of site I

Three thermodynamic parameters characterizing the binding of a protein ligand to a polynucleotide lattice can be obtained from titration data. These are K, the intrinsic binding constant; n, the site size (number of nucleotide residues occluded by one protein) ; and w, the parameter reflecting ligand-ligand binding co-operativity. In principle all three parameters can be extracted from a Scatchard plot of the binding data.

BINDING

SITE

I

OF

RIROSOMAL

PROTEIN

Sl

325

A typical fluorescence quenching binding plot (for poly(dC)) is shown in the Scatchard (1949) representation in Figure 1; here all the quenching is due to binding to site 1; since site II (Draper et al., 1977 ; and accompanying paper) shows little or no affinity for DNA polymers. Very similar plots were obtained with poly(dA). The observed curvature (lower calculated curve) is all attributable to overlap binding (McGhee & von Hippel, 1974); we conclude from the good fit of the data to this curve that w = 1 for the binding of single-stranded DNA polynucleotides to 81 protein, and therefore that this binding is non-co-operative. Even a very small degree of co-operativity (e.g. w = 5; upper calculated curve) gives a pronounced “hump” in a Scatchard plot such as Figure 1 (McGhee & von Hippel, 1974) and is not compatible with the experimental data.

FIG. 1. Scatchard plot of poly(dC) binding to Sl protein. The lower with the parameters K = 6.0 x IO6 Mm1 (measured in 0.1 x/standard size) = 5.3. For the upper calculated curve a co-operativity parameter

(solid) curve was calculated buffer) and m (occluded site (w) of 5 has been introduced.

The intrinsic binding constant for site I of Sl protein to polynucleotides can also be derived from plots such as Figure 1. Values of K (and other parameters) obtained from a number of titrations of Xl with poly(dA) and poly(dC) are summarized in Table 1. Within the limits of error the binding constants for these two deoxyribopolynucleotides are about the same. Table 1 also shows that the average site size (n) for these polynucleotides is about five nucleotide residues. We also carried out measurements to determine whether site I (or site II) has any affinity for double-stranded polynucleotides. For this purpose we used a sucrose gradient hand sedimentation technique which measures equilibrium binding constants (Draper et al.: 1977 ; Draper & von Hippel, manuscript in preparation). Typical results are shown in Figure 2. We find that a very high concentration of native phage X DNA is needed to displace Xl from the top of the gradient. From these data we estimate that the intrinsic binding constant for Xl protein to native DNA is -2X103 M-l (in 0.1 @tandard buffer). Thus site I prefers single-stranded polynucleotides by at least three orders of magnitude in the binding constant. Sl should therefore function as a “melting”

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TABLE 1 fhnmary

of site I polynucleotide K (M-I)

n (nucleotide

1.7&0*5X

1oq

6.0&2.0x

106§

binding parameters for X1 protein? w

m’ (ion-pairs)

-111

2.1*0.1/l

residues)

5.1&1.01/

t K, m, and w represent average values determined from Scatchard plots data; m’ was determined from the ionic strength dependence of K. $ For poly(dA) in 0.1 M/standard buffer. 5 For poly(dC) in 0.1 m/standard buffer. 11Average values for poly(dA) and poly(dC) in 0.1 M/standard buffer.

of fluorescence

titration

(double-helix destabilizing) protein, shifting the helix-coil transition of a base-paired sequence in favor of the coil form at equilibrium. In support of this notion, Szer et al. (1976) have shown that Sl protein can stabilize a mixture of poly(dA) and poly(dT) against renaturation at temperatures below the melting temperature of the double-stranded complex, and can induce the melting of base-paired hairpin loops in heated and retooled calf thymus DNA.

I

5

15

25

Fraction no FIG. 2. Band sedimentation of Sl and native DNA in a sucrose gradient. (65 mm) (--O--O--) and 3H-labeled Sl protein ( - 1O-9 M) (-e-e-) standard buffer were sedimented for 2 h at 35,000 revs/min jin a SW 50.1 gradient is 5% to 20% in 0.1 M/standard buffer. The top of the gradient is on

32P-labeled A DNA in 0.2 ml of 0.1 NI/ rotor. The sucrose the right.

BINDING

SITE

I

OF

RIBOSOMAL

PROTEIN

Sl

327

(b) Xalt dependence of site I bindilzg The contribution of electrostatic interactions to the total free energy of binding can be explored by examining the dependence of the apparent binding constant on salt concentration. Recently Record et al. (1976) have shown that such data can be interpreted to determine the number of basic amino acid residue-polynucleotide phosphate charge-charge interactions involved in stabilizing a nucleic acid-protein complex. In free nucleic acids the backbone phosphates are substantially neutralized by counterions; the fraction of each phosphate charge neutralized by thermodynamically “bound” counterions, 4, can be calculated. For a binding protein forming m’ ion pairs with polynucleotide phosphates, rn’# counterions will be displaced into solution : protein

+ polynucleotide

= complex

Record et al. (1976) demonstrated that the dependence constant, K, on the monovalent ion concentration ([M+])

f

m’#

M+.

(1)

of the apparent association may be written:

Thus the number of phosphates interacting electrostatically with a binding protein (m’) can be easily calculated. Two points should be emphasized with regard to the application of equation (2) to binding data. First, the predicted salt dependence of binding applies only to monovalent salts in the absence of multivalent cations. Added Mg’+, for instance, will bind tightly to the polynucleotide backbone, displacing monovalent cations and considerably complicating analysis of the electrostatic component of protein binding (Record et al., 1977). Furthermore, equation (1) may not adequately describe the It is possible that anions bound to the protein binding of a protein to a polynucleotide. are also released upon complex formation, thus complicating the interpretation of the slope of a plot of log K versus lOg[M+]. Record et al. (1976) have shown that anion binding is not involved for oligolysine binding to double-stranded DNA. For present purposes we assume that anions are not displaced by the binding of polynucleotides to site I (however, see the following discussion of possible anion binding effects involved in the quenching of Xl protein fluorescence by iodide ion). Figure 3 shows a plot of log K versus log[Na+] for the interaction of poly(dA) with site 1 of Sl. The slope of the straight line obtained is -1.63 ; using # = 0.78 (determined by poly(rA); Record et aZ., 1976), a value of m’ of 2.1kO.l phosphates per Sl binding reaction may be calculated (Table 1). Data for the binding of the oligomer d(pA), to site I (see below) are also included in Figure 3; the measured slope for this interaction is -1.43, less steep than that found with poly(dA). The theory of counterion binding upon which Record et al. (1976) based their analysis of the salt dependence of protein-nucleic acid binding assumes an infinitely long charged lattice. The average 4 value for oligonucleotides will be smaller than that of polynucleotides, since phosphates at the end of the chains will exhibit a lower charge density than those in the middle, and thus will bind fewer counterions per phosphate. Thus the apparent salt dependence for protein binding to oligonucleotides will be smaller than for polynucleotides, in agreement with our observations with d(pA), and poly(dA). A correction factor for the terminal .phosphates can

328

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[No+]

H,

VON

HIPPEL

(M)

FIG. 3. Dependence of log K on log [Na+] for d(pA), and poly(dA). Binding constants are calculated from fluorescence titrations of Sl in standard buffer with various concentrations of N&l. For the indicated titration of d(pA),, 5 miw-MgCl, was added to 0.1 Ivr/standard buffer. The slopes of the lines shown are -1.63 (for poly(dA)) and -1.43 (for d(pA),). (0) Poly(dA); (0) @A),; (A) d(pA), +5 mM-Mg2+.

be introduced to obtain z,&“,~, the average $ value for each phosphaOe of an oligomer X bases long; a value for this correction factor has been empirically determined from the dependence of melting temperatures on oligonucleotide length by Record & Lohman (1978). The predicted $J,” value for d(pA), is 0.71, which together with the determined value of m’#,, = l-43, gives m’ = 2.0. The data for poly(dA) and d(pA), binding to Sl are thus in good agreement, and imply that d(pA), must be binding in the entire site I binding site, at least to the extent to which the site is defined by electrostatic interactions. This analysis of the dependence of the apparent binding constant on salt concentration can be extended further to estimate the total non-electrostatic contributions to the binding interaction. For these purposes the binding free energy is partitioned into three components: a non-electrostatic term, a charge-charge interaction term, and a favorable entropy term due to the release of the bound counterions displaced by the protein ligand. In high salt concentrations the last term will be negligible. Record et al. (1976) have estimated the (unfavorable) charge-charge interaction free energy for the formation of a DNA phosphate-lysine complex in 1 M-Nacl (the NaCl standard state) as $0.18 kcal/mol. Extrapolating the data of Figure 3 to 1 M-Na+, and subtracting this free energy for the formation of two charge-charge interactions between the polynucleotide and the protein (we assume for this purpose that the proteinnucleic acid ion pairs have the same non-electrostatic free energy as a lysine-phosphate pair), we obtain a substantial favorable non-electrostatic free energy of N -6.7 kcal/mol. Since the nucleotide bases seem to interact very little with site I (see below), we assume that this free energy derives mostly from favorable protein contacts with the sugar moieties of the polynucleotide chain.

BINDING

SITE

I

OF

RIBOSOMAL

PROTEIN

Sl

329

Since many experiments done with Sl and ribosomes must be carried out in the presence of Mg2 + , we have determined whether the presence of this ion has any unusual effects on site I binding. Mg 2 + is a much more efficient neutralizer of nucleic a,cid phosphate charges than are monovalent cations ; it will displace “condensed” monovalent counterions from the nucleic acid backbone, and high concentrations of monovalent cations are required to displace it (Record et al., 1977). Therefore we expect a more substantial effect for Mg’+ on the electrostatic component of binding than would be predicted merely from the increase in buffer ionic strength, and this is indeed seen : adding 5 mM-Mg 2+ to 0.1 M/standard buffer reduces the binding constant of d(pA), by about a factor of five (Fig. 3). (c) Binding

of mono- and oligonucleotides

to site I

In order to gain some insight into the detailed structure of site I, we have studied the binding of a series of deoxyribose-containing mono- and oligonucleotides to this site. The binding constants determined by fluorescence quenching are shown in Table 2 ; a typical double reciprocal plot of a titration is shown in Figure 4. All binding constants shown are calculated assuming one binding site per protein?. Only in t,he case of ligands with affinities greater than lo6 M-I can the possibility of two binding sites be excluded by direct analysis of the data. The maximum quenching for all compounds listed is in the range 35 to 45’+& comparable to the quenching seen with poly(dA) and poly(dC) (Draper et al.; 1977). This holds true even for the small compound 2’-deoxyribose 5’-phosphate, which lacks a base altogether. Quenching of intrinsic protein fluorescence may be caused by charged or polar groups coming close to fluorescent tryptophans, either as part of the ligand or as a consequence of a ligand-induced conformational change in the protein. The phosphate anion is a likely quenching component of the nucleotide ligand; Kelly & von Hippel (1976) have shown that phosphate-quenching of protein tryptophan fluorescence is operative in the interaction of T4-coded gene 32 protein with single-stranded nucleotide oligomers. The fact that 2’-deoxyribose 5’-phosphate quenches as well as DNA oligomers and polymers means that, to a first approximation, it binds in the same way as the longer, base-containing compounds, either by orienting similarly in the binding site or by inducing a similar conformational change in the protein. An interesting feature in the data of Table 2 is the small change in binding affinities in going from 2’-deoxyribose 5’-phosphate to dAMP and dCMP. Evidently the addition of a. base onto the sugar-phosphate moiety has a negligible effect on the binding free energy, implying that most of the free energy of site I binding comes from interaction with the sugar-phosphate backbone. A comparison of d(pA),, d(pC),, and d(pT), affinities shows that site I binding indeed has little dependence on base composition. Another feature of Table 2 to be noted is that d(ApA) binds to two sites on Sl protein, as shown by the titration curve in Figure 5. The dimer binds stoichiometrically t The calculations also assume that site I binds oligomers in only one orientation, i.e. that the site is polar and discriminates between oligonucleotides binding in the 3’ + 5’ and 5’ + 3’ directions. Although one can conceive of a binding site able to bind oligonucleotides in either orientation, such a property is probably uncommon and has never been demonstrated. Available evidence indicates that the T4 gene 32 protein (Kelly 85 van Hippel, 1976) and ribonuclease T, (Walz & Terenna, 1976) have polar binding sites. If site I binding is nol polar, the binding constants calculated in Tables 1, 2 and 4 will be too large by a factor of two.

330

D. E. DRAPER

AND

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TABLE

Afinity

of Xl protein

HIPPEL

2

site I for mono- and oligodeoxyribonucleotides

Compoundt 2’.deoxyribose dAMP dCMP d&A)

VON

K ( x IO-6 m-1)$

5’.phosphate

§

d(pA)z d(pA), d(pA)a 4pTh d(P&

0.11 0.19 0.082 > IO.0 1.9 3.1 4.4 1.4 O-8 2.7

10.01 ho.01 i: 0.02 *o+! 10.2 kO.5 50.1 -+O.l ho.2

t Abbreviations are defined in Materials and Methods. $ All binding constants are calculated assuming one binding site per protein; errors reported are 1 S.D. § Titrations with this compound did not fit a single site binding isotherm, but required tho assumption of 2 binding sites of different affinities. See text for discussion.

0

I

I

I

I

I.0

2.0

3.0

4.0

IdAMP]-’

FIG. 4. Double reciprocal O.lM/standard buffer. From Qnlax = 0.41.

x 80

5

plot of the binding of dAMP to Sl protein. Sl is 0.91 x 10-s M in the least-squares line fitted to the points, K = 1.9~ 105 M-I and

in the first part of the curve, requiring a binding constant of at least lo7 M-l; this is followed by a second, weaker binding with KN 1.9 x lo5 M-l. This finding could reflect d(ApA) binding in both site I and the RNA-specific site II (discussed in the accompanying paper), if the ribose-deoxyribose discriminatory ability of site II functions only with longer oligomers and polymers, while short oligomers bind in some portion of the binding site which is indiscriminate. This possibility is ruled out by the following experiment. Xl is first titrated with the oligomer d(pA),, which binds detectably to only one site on Sl, and then with d(ApA). If any binding of d(ApA) to site II occurs, an additional quenching should be observed (as is the case with the oligoribonucleotide r(A(pA),), discussed in the accompanying paper). Figure 6 shows that this is not the case ; very little additional

BINDING

SITE

I OF RlBOSOMAL

[d(ApA)]

FIG. 5. Fluorescence titration of Sl with d(ApA). The curved portion of the solid line is a binding and Q,,, = 0.20.

PROTEIN

Sl

331

I/L’M)

Sl was 0.77 x 1Om6M in 0.1 ~~/standard buffer. isotherm calculated using K = 1.7 x IO5 x-l

quenching is seen, and it can be accounted for entirely by d(ApA) binding to the fraction of site I not saturated already by d(pA),. These results suggest that there are two subsites within site I, arranged so that each can interact independently with a d(ApA) molecule, or so that both can bind simultaneously to a longer oligo- or polynucleotide. A schematic illustration of how these subsites might be arranged within site I is shown in Figure 8; since additional data bearing on the structure of site I will be presented, a full consideration of subsite geometry will be deferred to the Discussion. Table 2 also shows that the binding constants for dinucleotides and longer oligomers are comparable to the intrinsic binding constants measured with polynucleotides

0.30

d(ApAi

0.20

-

0

0

I

2

3 [Oligomer]

4

5

6

(p)

FIG. 6. Fluorescence titration of Xl with d(pA), followed by d(ApA). Sl (0.73 x 10m6 NI in 0.1 M/standard buffer) was titrated with d(pA)* (0) to 3.12 x 1O-6 M, and then with d(ApA) (0) (arrow). The bindmg cww drawn is fitted t.o the am tit.ration, with K = 1-2 x lo6 ?.I-' and

Q,,, = 0.29.

332

D.

E. DRAPER

AND

I’.

H.

VOX

HIPPEL

(Table 1). Since the apparent binding constant for oligomers just long enough to cover the functional groups of the protein should be equal to K, while that for a long polymer should be Kw, we conclude, in accord with the evidence of Figure 1, that site I binding is non-co-operative (W ‘v 1). (d) Iodide quenching

of site I fluorescence

The exposure of protein fluorophores to solvent can be studied by “solute perturbation” methods. The excited state of a fluorophore can be deactivated by fluorescence, various non-fluorescent energy t’ransfer mechanisms, and collisional quenching by a diffusible molecule. For an ideal collisional quenching process, the dependence of the quantum yield of a fluorophore on the concentration of quencher is given by the Stern & Volmer (1919) relation:

PO/F = 1 + KpLQl,

(3)

where E”/B is the ratio of the unquenched to quenched Auorophore quantum yield, and [Q] is the concentration of diffusible quencher. K,, the “quenching constant”, is composed of the rate constants for the different competing pathways of deactivation of the fluorophore excited state, and will depend on the quantum efficiency of the fluorophore, the diffusion coefficients of the quencher and the fluorophore, and the accessibility of the fluorophore to the quenching molecule. A plot of PaIF versus Q should be linear, with a slope of K,. The iodide anion, I-, has been shown by Lehrer (1971) to quench tryptophan by a collisional mechanism: K, shous the expected dependence on temperature and viscosity for a diffusion controlled process, and tryptophans in lysozyme which are on the outside of the protein or in the binding site are more easily quenched than those buried in the protein interior. It should be noted, however, that quenching of protein tryptophans by iodide will rarely proceed by a strictly collisional mechanism. Any charged groups on the surface of a protein will gather a cloud of counterions, influencing the diffusion of iodide in the vicinity of exposed tryptophans. Data plotted according to equation (3) will still be linear if iodide is weakly bound or repelled by the protein; however: K, will then be dependent on ionic strength, as shown by Lehrer (1971) with charged tryptophan derivatives. In 0.1 M/standard buffer, the model compound N-acetyl tryptophanamide, which should resemble a protein tryptophan maximally exposed to solvent? has a K, va,lue of 10.7 M-l. This is in good agreement with the value of 12.0 Me1 obtained by Lehrer (1971) for this compound, taking into account the increase in viscosity due to the loo/, glycerol in our buffer. K, for Sl protein under the sa,me conditions is 9.9 IM-~ (Table 3 and Fig. 7). This large quenching constant for X1 indicates that quenching is clearly not proceeding by a purely collisional mechanism. For a fully exposed fluorophore K, is proportional to the sum of the diffusion coefficients of the quencher and fluorophore (Forster, 1951). Since the diffusion coefficient of a protein will be negligible relative to the diffusion coefficient of I-, the maximum possible quenching constant for a protein tryptophan will be a.bout SO?/, of that for N-acetyl tryptoFurthermore: the wavelength of the tryptophanamide (Lehrer, 1971), i.e. -841~~. phan fluorescence peak of Xl is shifted appreciably toward shorter wavelengths relative to that of N-acetyl tryptophanamide (Draper et cnl., 1977). This is usually taken to mean that the fluorophores are at lea,st partially “buried” in an environment with an average polarity appreciably less than water, and is clearly not

BINDING

SITE

I

OF

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TABLE

PROTEIN

Sl

333

3

Iodide quenching of intrinsic protein Juorescence of free Xl protein and Xl-oligonucleotide corrplexes Oligonucleotide

bound

K,

None (0.1 nI/standard buffer) None (0.33 ~~/standard buffer) d(ApA) (1 bound/protein) d(ApA) (2 bound/prot,ein)

9.9*0.2 8.3*0.2 7.1~kO.1 4.5t0.2 7.6hO.l 4.6kO.l 4.3kO.2 24.51,tO.l

d(pAh 4rWs pOly(W WM + Both

site I and site II

(M - ‘)

sakwated.

[KI] (~1 Fx. 7. Iodide quenching of Sl protein. Sl was titrated with potassium iodide in either 0.1 RI (A) or 0.33 M/standard buffer (C) and the data plotted according to t,he Stern-Volmer equat,ion. The slopes of the lines in the 2 buffers are K, = 9.9 and 8.3 iv-l, respectively.

compatible with an iodide quenching constant of this magnitude if quenching is purely collisional in nature. The most likely explanation for this large quenching const)ant is that Xl binds anions weakly in the vicinity of one or more protein tryptophans, thus increasing the effective concentration of iodide nea.r a tryptophan. Since nucleic acid binding proteins must, carry a patch of positive charge if they are to interact electrostatically with nucleic acid, weak anion binding by the protein in the vicinity of nucleic acid binding sites might’ be expectedt. K, for Xl at higher salt concentrations (0.33 M/standard buffer) is decreased to -8.3 M-l (Table 3 and Fig. ‘i), while K, for N-acetyl tryptophanamide ? This conclusion appears contrary to our assumption of no anion binding in interpreting the salt dependence of the apparent binding constant of Sl to deoxyribonucleotides (above). In order for bot,h conclusions to be congruent we must assume that the anion binding which accounts for the enhanced I- quenching is either not strong enough to reduce measurably the effective charge density of the positively charged protein binding domain, or else that these anions are not displaced by complex formation with nucleic acids. It is also certainly possible that our assumption of no amon binding is incorrect, and that the actual value of m’ is therefore somewhat less than the value of 2.1 ion pairs per Sl interaction listed in Table 1.

334

D.

E.

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is virtually unaffected (data not shown). This confirms the existence of a weak anion binding site near a tryptophan on Xl; higher chloride concentrations presumably compete with iodide for this binding site and thus decrease the quenching observed. The effects on the quenching constant of various oligomers bound to Xl are summarized in Table 3. When both site I and site II are saturated with (rC),, K, is reduced to 2.6 M-l, a reasonable value for collisional quenching of tryptophans relatively inaccessible to solvent. Presumably, oligonucleotide binding has neutralized anion binding sites on Sl and/or quenched surface tryptophans. The series of deoxyadenylate oligomers fall into two distinct classes: those which result in K, N 4.5 M-l: and those which yield K, p 7.4 M-l. The lower quenching constant is reasonable for a collisionally quenched protein tryptophan, and may represent a complete neutralization of protein anion binding sites by the bound polynucleotides. A K, value of 7.4 Mu1 is perhaps possible for a collisionally quenched tryptopha,n which is on the protein surface and fully accessible to solvent, but since Xl tryptophans are on the average in a somewhat hydrophobic environment (Draper et al., 1977), this value more likely reflects some remaining anion binding by Xl. This iodide quenching data can be understood in terms of the two site I subsites hypothesized from the d(ApA) binding data. If each subsite has some positive charge which weakly binds anions and is neutralized by interact.ion with bound nucleotide phosphate groups (with one or more protein tryptophans in the vicinity of the subsites), binding nucleotides in the subsites should then reduce the iodide quenching in two stages, depending on whether one or two anion binding sites are neutralized. This is indeed the behavior seen: one d(ApA) bound reduces K, to 7.1 M-l, while a second d(ApA) bound gives a K, value of 4.5 M- l. Poly(dA), which must bind in both subsites, reduces K, to about the same value as is achieved by the binding of two d(ApA) molecules. The data suggest that the longer oligomer d(pA), binds in both subsites (K, = 4.6 M-l), while the shorter oligomer d(pA), binds in only one subsite (KQ = 7.6 M-l). (e) InJluence of site II binding

on site I

We must also consider whether there is any effect of binding an oligomer in site II on the binding constants measured for site I. The RPU’A oligomer (rC)z is bound tightly and co-operatively by site II, and titrates site II nearly completely before binding in site I (Draper et al.: 1977). We th erefore titrated site II with this oligomer, and then site I with several of the oligomers already tested. The results are shown in Table 4. The most dramatic effect is on the binding of d(ApA); the data obtained TABLE 4 Binding

constants of XI protein presence Oligonucleotide~

density

in the

K(XlO-“M-l)

dAMP WpA) d(pA), d(p& t Sl was first titrated to a binding with the indicated deoxyribonucleotide.

to deoxynucleotides

of (rC))20

0.35*0.04 0~50+0~03 0.34*0.03 0.50+0.04 of 2

81 molecules

per oligomer

with

(rC),,

and then

BINDING

SITE

I OF RIBOSOMAL

PROTEIN

Sl

335

cannot be used to distinguish between two binding subsites of equal affinity and one remaining subsite, but clearly the strong binding (R > 1V RI-~) observed when site II is unoccupied has been abolished. The affinities of d(pA), and d(pA)s are also significantly reduced when site II is bound, by factors of roughly ten and three, respectively. Thus site II binding has a distinct weakening effect on the binding affinity of site I; t,he principle of microscopic reversibility requires t.hat site T has a similar effect, on site II binding.

4. Discussion (a) Site I topography We have presented evidence in this paper that Sl site I has two distinguishable “subsites” which are involved in nucleic acid binding. Three sets of experiments lead to this conclusion: (1) two d(ApA) molecules can bind in site I ; larger molecules of only one molecule per protein; (2) there (e.g. d(pA),) bind with a stoichiometry are two classes of (iodide) quenching constants for site I-oligonucleotide complexes which differ depending on whether one or two d(ApA) molecules are bound, and on whether a long or short oligomer is bound; (3) based on the salt-dependence of the apparent binding constant, site I forms about two ion pairs with DNA phosphates. These observations are summarized in the model shown in Figure 8.

L

FIG. 8. Schematic diagram of site I binding topography. Two d(ApA) binding in the two site I subsites. See text for further explanation.

molecules

are shown

Each subsite is shown containing a positively charged protein residue which can form a charge-charge complex with a DNA phosphate. One or more protein tryptophans are positioned suitably for quenching by bound nucleotides. The spacing between the subsites is limited by two considerations: (1) the distance between the phosphate interaction sites cannot be greater than the site size determined from polymer titrations (i.e. 51&1*0 nucleotide residues); (2) molecules larger than d(ApA) cannot bind in both subsites simultaneously. Both observations suggest a close spacing of the subsites. If the total binding free energy of site I for a polynucleotide is calculated by adding the free energies of binding two d(ApA) molecules in the two subsites (including a

336

D. E. DRAPER

AND

P. H.

VON

HIPPEL

cratic term to take into account the fact that two moles of ligand are bound), the result grossly overestimates the polynucleotide affinity; we calculate more than -17 kcal/mol, versu.s only -86 kcal/mol observed with poly(dA). Therefore there must be some unfavorable interactions in the site which d(ApA) can avoid, but which a longer polynucleotide cannot : for example; a part of the molecule may be constrained into an unfavorable conformation, or some part of the chain may be put into an unfavorable (e.g. negatively charged or hydrophobic) environment. The presence of unfavorable as well as favorable interactions in a protein binding site may help to explain an anomaly in the oligomer binding data of Table 2. B simple view of protein-nucleic acid interactions predicts that binding affinities should increase with increasing chain length of oligomers, both because more interactions with the binding site become possible, and because more potential binding sites become available (i.e. there are more ways of arranging a binding protein on a larger oligomer). Yet d(pA),, for instance, binds in sit’e I about threefold more tightly than d(pAh> suggesting that a shorter oligomer can “fit” into site I with less loss of conformational free energy or fewer unfavorable interactions than longer oligomers. Other nucleic acid binding sites show a similar trend: e.g. d(pT), binds a factor of 60 more tightly to Staphylococcal nuclease than does d(pT), (Cuatrecasa,s et al.: 1968), and d(pA), binds a factor of two better to T4 gene 32 protein than d(pA), (Kelly et al., 1976). It appears that detailed understanding of the structure of nucleic acid binding sites will require consideration of unfavorable, as well as favorable, contributions to the free energy of binding. (b) Xpeci$city Three conclusions can be drawn nucleotides recognized by site I.

about

of site I binding the features

and conformations

of poly-

(1) The binding free energy of site I derives almost entirely from interaction with the sugar-phosphate backbone of the polynucleotide. We have been unable to detect any significant’ dependence of site I binding on base composition, and chemical modification of polynucleotide bases with either formaldehyde (accompanying paper) or chloroacetaldehyde (see below) has no effect on binding affinity. (2) Binding in site I does not require major conforma’tion changes in the polynucleotide. Szer et al. (1976) report finding no hyperchromic changes as a consequence of binding either poly(dA) (a highly stacked polymer) or poly(dT) (almost entirely unstacked) to Sl. We have made fluorescent adenine derivatives of d(pA),, d(pA), and denatured calf thymus DNA, using the chloroacetaldehyde reaction described by Secrist et al. (1972). The fluorescence of this etheno-adenine is quenched by about a, factor of ten in stacked dinucleotide phosphates (Tolman et al., 1974) and polynucleotides (our unpublished results), and is therefore highly sensitive to the relative conformation of the bases. Although Sl binds these derivatized compounds with the same affinity as the unmodified materials, no significant effect is seen on the ethenoadenine fluorescence. (3) Binding is highly specific for single-stranded polynucleotides relative to double stranded forms. In all three respects site I binding resembles closely the binding of ribonuclease to DNA (Jensen & von Hippel, 1976). RNase binding is also non-co-operative; furthermore, RNase can denature native DNA well below its normal melting temperature,

BINDING

SITE

I OF RIBOSOMAL

PROTEIN

Sl

337

suggesting that’ site I may also show “melting protein” activity. Although Szer et al. (1976) found that Sl could melt out hairpins in calf thymus DNA below their normal T, value, a detailed study of the degree to which site I can lower melting temperat,ures, and of how extensive a base-paired structure can be melted, has not yet been performed. The ability of site I to melt out double-helical regions in nucleic acids may well be related to its role in vivo; it could, for instance, aid the initial binding or the movement of the ribosome along the messenger RP\TA by melting out double-helical hairpin loops in the message. The possible functional significance of the site I properties we have determined here will be discussed further in the accompanying paper. This research was supported in part by United States Public Health Service research grants GM-15792 and GM-15423, and a Predoctoral Traineeship (to D. E. D.) from a United States Public Health Service training grant GM-00444. This work has been submitted (by D. E. D.) to the University of Oregon Graduate School in partial fulfillment of the requirements for the Ph.D. We are indebted to Mrs Ruth Draper for expert technical assistance with many phases of this work. REFERENCES

16, 4757-4768. Butler, A. P., Kevzin, A. & von Hippel, P. H. (1977). Biochemistry, Cuatrecasas, P., Wilcheck, M. & Anfinsen, C. (1968). Science, 162, 1491-1493. Dahlberg, A. & Dahlberg, J. (1975). Proc. Nut. Acad. Sci., U.S.A. 72, 2940-2944. Draper, D. E. & von Hippel, P. H. (1976). In Molecular Mechanisms in the Control of Gene Expression (Nierlich, D. P., Rutter, W. J. & Fox, C. F., eds), pp. 421-426, Academic Press, New York. Draper, D. E. & von Hippel, P. H. (1978). J. Mol. Biol. 122, 341-361. Draper, D. E., Pratt, C. W. & von Hippel, P. H. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 4786-4790. Forster, T. (1951). Fluorescence Orgalzischer Verbin,dungen, Vandenloch and Rupnecht,, Gottingen. Jenik, B. (197 1). Physicochemical Characteristics of Oligonucleotides and Polynucleotides, IFI/Plenum, New York. Jensen, D. E. & von Hippel, P. H. (1976). J. Biol. Chem. 251, 71987214. Jensen, D. E., Kelly, R. C. & von Hippel, P. H. (1976). J. Biol. Chem. 251, 7215-7228. Kao-Huang, Y., Revzin, A., Butler, A. P., O’Connor, P., Noble, D. & von Hippel, P. H. (1977). Proc. Nat. Acad. Xci., U.S.A. 74, 4228-4332. Kelly, R. & von Hippel, P. H. (1976). J. Biol. Chem. 251, 7229-7239. Kelly, R. C., Jensen, D. E. & von Hippel, P. H. (1976). J. Biol. Chem. 251, 7240-7250. Klotz, I. M. & Hunston, 0. L. (1971). Biochemistry, 10, 3065-3069. Langhrea, M. & Moore, P. B. (1977). J. Mol. Biol. 112, 399~-422. 10, 3254-3263. Lehrer, S. S. (1971). Biochemistry, Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193,

265-275. McGhee, J. Record, M. Record, M. Record, M. Scatchard, Secrist’, J.

D. T., T., T., G. A.,

& von Hippel, P. H. (1974). J. Mol. Biol. 86, 469-489. Jr & Lohman, T. M. (1978). Biopolymers, 17, 159-166.. Jr, Lohman, T. M. & de Haseth, P. L. (1976). J. ll/loZ. Bioi. 107, 145-158. ,Jr, de Haseth, P. L. & Lohman, T. M. (1977). Biochemistry, 16, 4791~. 4796. (1949). Ann. N.Y. Acad. Sci. 51, 660-672. Barrio, J. R., Leonard, N. J. & Weber, Cr. (1972). Biochemistry, 11,

3499-3506. Shine, J. & Dalgarno, L. (1974). Proc. Nat. Acad. Sci., U.S.-3. 71, 1342--1346. Steitz, J. A., Wahba, A. J., Laughrea, M. & Moore, P. B. (1977). 1VucZ. acids Res. 4, l-15. Stern, 0. Xs Volmer, M. (1919). Phys. 2. 20, 183-193. Szer, W., Hermoso, J. M. & Boublik, M. (1976). Biochem. Biophys. Res. Commun. 70, 957-964.

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M., Aviram, M., Kanarek, A. & Weiss, A. (1972). Biochim. Biophys. Acta, 281, 381-392. Tolman, G. L., Barrio, J. R. & Leonard, N. J. (1974). Biochemistry, 13, 4829-4878. T’so, P. 0. P. (1974). In Basic Principles in Nucleic Acid Chemistry (T’so, P. 0. P., ad.), vol. 2, pp. 3066470, Academic Press, New York. van Dieijen, G., Vanderlaken, C., van Knippenberg, P. & van Duin, J. (1975). J. Mol. Biol. 93, 351-366. van Dieijen, G., van Knippenberg, P. H. & van Duin, J. (1976). Eur. J. Biochem. 64, 511-518. van Duin, J. & van Knippenberg, P. H. (1974). J. Mol. Biol. 84, 185-195. Walz, F. & Terenna, B. (1976). Biochemistry, 15, 2837-2842.