Proc. Nati. Acad. Sci. USA Vol. 76, No. 3, pp. 1040-1044, March 1979 Biochemistry Interaction of ribosomal S1 with (SI polynucleotide binding sites/30S ribosomal subunits/S1-30S binding constants/S1-30S binding stoichiometry/ 16S ribosomal RNA) DAVID E. DRAPER* AND PETER H. VON HIPPEL Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403 Contributed bt Peter H. von Hippel, November 16,1978

ABSTRACT The binding affinity of Escherichia coli ribo- tein to ribosomes and to ribosomal subunits and report here the somal protein SI for 30S ribosomal particles has been deter- results of some preliminary attempts at a quantitation of the mined by a sucrose gradient band sedimentation technique; the association constant (K) for the binding of one SI protein per Si- binding interaction. active 30S ribosomal subunit is t2 X 108 M-1. The involvement Three basic questions are addressed: (i) What is the binding of the two polynucleotide binding sites of SI protein (site I affinity of S1 for the 30S (and 50S and 70S) ribosomal compo- binding single-stranded DNA or RNA, and site II binding sin- nents? (ii) Does either S1 polynucleotide binding site contribute gle-stranded RNA only) in the SI-ribosomal interaction have been examined by competition experiments with polynucleo- to the Sl-ribosome interaction, or is this binding entirely at- tides of known affinity for the two sites. We find that site I does tributable to interactions with other of the 30S particle? not contribute to the interaction; site II binding appears to (iii) Is either S1 polynucleotide binding site available for in- provide a major part of the binding free energy, presumably by teraction with mRNA when S1 is bound to the ribosome? We interaction of SI with the 16S rRNA of the 30S particle. The re- maining binding free energy is probably derived from the in- conclude from the evidence presented here that most of the free teraction of SI protein with other proteins of the 30S subunit. energy of the protein S1-30S ribosomal subunit interaction is The affinity of SI for 70S ribosomes is about the same as that derived from site II binding to 16S rRNA, and that site I may for the 30S subunit; the affinity of SI for 50S subunits is much thus be available to facilitate mRNA binding to the ribo- less. Binding affinities and stoichiometries of SI protein with some. "inactive" 30S ribosomal subunits have also been examined. Studies from several laboratories have suggested that Esche- MATERIALS AND METHODS richia coil Si is involved in mRNA binding to the ribosome during initiation of protein synthesis. Si has Buffers. Experiments involving ribosomes or ribosomal been shown to be required for efficient binding and subunits in the active conformation (9) were performed in 5 of poly(rU) and phage MS2 RNA (1), and numerous protection mM MgSO4/100 mM NH4Cl/20 mM Tris, pH 7.7/3 mM 2- studies (summarized in ref. 2) have implicated this protein as mercaptoethanol (buffer A). Experiments with "inactive" ri- part of the ribosomal mRNA binding site. It has been known bosomes were carried out in the same buffer, except that the for some time that S1 can bind to RNA, and this binding has MgSO4 concentration was 0.3 mM (buffer I). been assumed to be responsible, in some way, for the influence Materials. Polyribonucleotides were purchased from Miles; of S1 on mRNA binding to the ribosome (3). polydeoxyribonucleotides were from Collaborative Research Recently, we demonstrated the presence of two distinct (Waltham, MA). S1 protein labeled with [3H]leucine (New polynucleotide binding sites on Si protein (4). Site I binds well England Nuclear) and unlabeled S1 were prepared from E. coli to single-stranded RNA or DNA, interacts mostly with the MRE 600 cells by DNA-cellulose chromatography as described sugar-phosphate backbone, and shows little dependence of (4, 5).t [3H]S1 and unlabeled S1 coelectrophoresed in sodium binding on base composition (5). In contrast, site II shows a high dodecyl sulfate/polyacrylamide gels and competed for binding degree of specificity for single-stranded RNA over DNA and to the 30S ribosomal subunit. "High-salt-washed" 30S ribosomal appears to interact primarily with the RNA bases and sugars subunits and 70S ribosomes were prepared from E. coli MRE rather than with the backbone phosphates (6). Although the intrinsic of site II for is also affinity polynucleotides relatively * Present address: Department of Molecular, Cellular, and Develop- independent of base composition, binding has been shown to mental Biology, University of Colorado, Boulder, CO 90309. be cooperative for the polyribopyrimidines tested, and non- t Our preparative procedure for Si protein differs from procedures cooperative for the polyribopurines, resulting in a net (coop- used by most other workers in that we use no urea, LiCl, or other erative) binding preference of 102 in the apparent binding denaturants. Therefore, to facilitate comparisons with the results of constant for polypyrimidines over (noncooperatively bound) others, we define Si (as prepared by our DNA-cellulose procedure) as follows (see also ref. 10). The protein prepared by our method polypurines (6). coelectrophoreses (in sodium dodecyl sulfate/polyacrylamide gels) Proposals have been put forward in the literature suggesting exactly with the largest protein obtained from 30S particles and with that the function of S1 in protein synthesis is to perturb the the slowest-migrating band of 30S protein electrophoresed at pH structure of the 16S rRNA (7) and also that S1 may melt the 4.5 in urea/polyacrylamide gels. It rebinds to 30S subunits stripped secondary or tertiary structures of mRNA during initiation (8). (>98%) of SI by low-salt treatment (3), as well as to well-washed 30S In light of these varying hypotheses regarding S1 function, as ribosomes that contain less than stoichiometric amounts of Si, re- well as the definition of the two very different polynucleotide sulting in an increase in the intensity of the Si band in gels of the proteins of the resulting 30S particles. Our Si preparation also binding sites on S1, we have examined the binding of this pro- stimulates poly(rU) binding to 30S subunits (1, 3). Preliminary ex- periments (R. Anderson and P. Hoben, unpublished results, this The publication costs of this article were defrayed in part by page laboratory) suggest that site I of the protein may be partially inacti- charge payment. This article must therefore be hereby marked "ad- vated by standard (high urea and LiCl concentrations) preparative vertisement" in accordance with 18 U. S. C. §1734 solely to indicate procedures, resulting in less effective binding of the protein to sin- this fact. gle-stranded deoxyribopolynucleotides and to DNA-cellulose. 1040 Downloaded by guest on September 24, 2021 Biochemistry: Draper and von Hippel Proc. Natl. Acad. Sc4. USA 76 (1979) 1041 600 cells by standard procedures (I1), and 30S subunits more still cosedimenting with the ligand at the end of the experiment than 98% depleted of SI protein were prepared by a low.-salt is thus'generally not the fraction bound prior to sedimentation treatment (3). The extent and specificity of depletion was (as is frequently, and erroneously, assumed); rather, a much judged by polyacrylamide gel electrophoresis in sodium dodecyl smaller amount of protein remains bound and the experiment sulfate at pH 8.1 and in urea at pH 4.5. All ribosomes were should be viewed as analogous to the multiple and sequential stored at -70°C in 3-fold concentrated buffer A (15 mM equilibria that apply in column chromatography, in which free MgSO4, etc.) in the activated state. Concentrations of SI and protein is progressively washed out of the complex (i.e., off the of ribosomal particles were calculated by using published ex- column) by successive aliquots of buffer. Given the distance of tinction coefficients (4, 11). sedimentation and original thickness of the band, the fraction Sucrose Gradient Band Sedimentation. Band sedimentation of binding protein still cosedimenting with the fast component, experiments were conducted and analyzed as described in detail and the concentration of binding sites on the rapidly sedi- elsewhere (12). All sedimentation experiments were carried out menting component, an association constant can be calculated with 5.0-ml 5-20% (wt/vol) sucrose (RNase-free grade, Sigma) (12). In general, conditions are arranged to provide a large gradients made up in the appropriate buffer. The initial bands initial excess of potential binding sites over binding protein, layered on the gradients were 0.2 ml in volume and generally although this is not required; in addition, if several classes of were 0.06-0.2 ,uM in ribosomal subunits and less than 0.1 nM binding sites of differing affinity are present, only those binding in [3H]S1 protein. Fraction volumes were 0.2 ml, and z25 most tightly will generally be measured quantitatively. fractions were collected dropwise by perforating the bottom of each nitrocellulose centrifuge tube after centrifugation. The experiments were conducted in a Beckman model L2-65B RESULTS preparative ultracentrifuge using a SW 50.1 swinging-bucket Association Constant of S1 Protein with 30S Ribosomal rotor. The total centrifugation time was adjusted so that the Subunits. Fig. 1 shows the sedimentation pattern of 30S sub- fastest sedimenting peak moved approximately one-third of units with [3H]SI protein. The subunits have been specifically the distance down the tube (it.5 hr at 40,000 rpm and 18°C depleted of SI by a low-salt treatment (3). Because it has been for 30S ribosomal subunits). shown that exposure to low salt concentrations puts 30S subunits Determination of Association Constants. Binding constants into an inactive (in terms of protein synthesis) conformation, were determined directly by sucrose gradient band sedimen- the subunits have been restored to the active form by warming tation. In essence, the method involves sedimentation, through in a high-salt buffer before use (9). a sucrose gradient, of a band of rapidly sedimenting ligand (e.g., An average association constant for SI binding to 30S subunits a ribosomal subunit or a large nucleic acid) mixed with a much (K30"81) of 1.7 X 108 M-1 has been calculated from several such more slowly sedimenting binding protein. The system is ar- sedimentation runs (Table 1), based on an assumed binding ranged so that the complex, together with free ligand, sediments stoichiometry of one tightly bound SI per SI-depleted 30S well ahead of unbound protein, thus reducing the concentration particle. This binding stoichiometry has been confirmed di- of total protein in the band. Sedimentation is slow compared rectly by a competition band sedimentation experiment in- to the rate of attainment of binding equilibrium; therefore, as volving [3H]S1, 30S ribosomal subunits, and a several-fold excess unbound protein is left behind, the complex dissociates to re- of unlabeled SI protein. The amount of SI protein sedimented establish binding equilibrium, and the newly dissociated protein out of the original band (and thus initially bound to the 30S is left behind in its turn.t The result at the end of the run is a ribosomal particle) was very close to 1.0 copy of SI per 30S ligand peak cosedimenting with a certain fraction of the original protein and a trailing smear of free protein that has been left behind further up in the gradient. The amount of protein found * These considerations must, of course, be modified if the half-life of dissociation of the complex becomes comparable to the rate of sedi- mentation of the band (12). For this system we assume that equili- bration is fast relative to the rate of sedimentation (the time required to sediment the ribosomal subunit peak by one bandwidth is greater than 10 min). The following arguments support this assumption. (i) If, as seems reasonable, the binding of SI to 30S subunits is assumed to be diffusion-controlled, the t1/2 for dissociation is smaller than 100 msec. Even if the binding rate were 2 orders of magnitude smaller .than diffusion-controlled, tl/24di. would still be smaller than 10 sec. (ii) Similar values of KSoSs are obtained by the sucrose gradient band sedimentation procedure and by polynucleotide competition experiments (see Tables 1 and 2); the latter determinations are based on fluorescence measurements made at equilibrium under "non- transport" conditions (5, 6). (iii) The measured values of Ksossj do not vary with speed of centrifugation (see Table 1). (iv) Laughrea and Moore (10) have demonstrated complete fast exchange (on a biochemical time scale) of free and 30S subunit-bound SI protein by various means. These experiments show that t1/2,dioc. must be less than 5-10 min. (v) Perhaps the strongest argument depends on 10 15 20 25 the fact that "tracer" quantities of Si (less than one SI molecule per Fraction 103 30S subunits) are used in these experiments (e.g., Figs. 1 and 2). FIG. 1. Sedimentation of 30S ribosomal subunits with [3HJS1 On this basis we would expect the sedimentation coefficient of the protein. Here, Si-depleted 30S subunits (at a concentration of0.1 ,4M) 30S particles, as reflected in the apparent sedimentation rate of the have been sedimented with [3H1S1 protein (total concentration <0.1 labeled Si, to be unperturbed by S1 binding if the rapid equilibration nM) on a sucrose gradient in buffer A; sedimentation is from right to assumption is valid. We find that the sedimentation rate of the 30S left. From the amount (58.2%) of the [3H]S1 estimated to remain with particles is indeed the same whether monitored directly or via the the 30S peak, a value of K30S-So 1.5 X 108 M-1 is calculated, as transport of labeled S1 protein. described in detail in ref. 12. Downloaded by guest on September 24, 2021 1042 Biochemistry: Draper and von Hippel Proc. Natl. Acad. Sci. USA 76 (1979) Table 1. Determination of protein Sl-30S ribosomal subunit binding constants by the band sedimentation method* Rotor speed, K30sl, Buffert rpm X 10-3 M-1 X 10-8t

A (3) 40 1.7 + 0.9 E zU A§ (4) 40 1.6 + 0.5 A(1) 35 1.9 0.6 0.

A(1) 30 1.5 0.4 - .I-15 1 (5) 40 6.0 ± 3.4 I (2) 30 5.0 ± 0.7 1(1) 22 5.7 ± 1.4 10 1 * All runs were at 18'C, except as noted. t Buffers are defined in Materials and Methods. The number of in- dependent determinations made of each binding constant is shown in parentheses. Shown ± SD. ~~~~I' § Run at 60C. 1'5 215 subunit. A stoichiometry of one Si bound per active 30S ribo- Fraction somal particle is also in accord with the results of Laughrea and FIG. 2. Competition between 30S subunits and polynucleotides for [3H]S1 binding. Here, S1-depleted 30S ribosomal subunits (at a Moore (10, 13) (see Discussion). concentration of 0.94 MM) have been sedimented in buffer A with Because binding interactions often involve changes in partial [3H]S1 protein (total concentration, <0.1 nM) and either 1.45 mM specific volume of the system [as manifested by a pressure- poly(rC) (0-0) or 1.36 mM denatured calf thymus DNA (0-0) dependent value of the binding constant (14)], we determined (polynucleotide concentrations expressed on a per nucleotide residue KSoSjs at several rotor speeds (Table 1); no speed-dependent basis). effects on the apparent association constant were observed. The suggestion that Si affects the secondary structure of a region fecting the Sl-ribosomal interaction. However, site II (RNA of the 16S rRNA (7) also led us to measure the Si-30S binding specific) is apparently involved in binding Si to the 30S sub- constant at a second temperature; any melting of rRNA ac- unit. companying SI binding should result in a variation with tem- Data from competition experiments, such as that shown in perature of the apparent Si affinity for 30S subunits. No sig- Fig. 2, together with the known affinities of site II for poly(rC) nificant difference was found in Ka3o-ss at 60C and at 18'C. and poly(rA) under various salt conditions (6), can be used to Polynucleotide-Si Protein Competition Experiments with calculate the absolute affinity of Si for 30S ribosomal subunits. 30S Ribosomal Subunits. The two polynucleotide binding sites The results are shown in Table 2 and are in good agreement of Si protein can be distinguished by their RNA-DNA binding with the values of Kos-sl determined by the sucrose gradient specificity; site I does not appear to discriminate between these band sedimentation procedure (Table 1). single-stranded polynucleotides, whereas site II is highly specific Competition for S1 Protein among 30S, 50S, and 70S Ri- for RNA (4). Thus, by setting up competitive binding experi- bosomal Particles. Competition experiments were also be ments in which Si is partitioned between 30S subunits and carried out to examine the affinity (relative to 30S subunits) of various polynucleotides, we can determine which (if either) of SI protein for 50S and 70S ribosomal particles. In buffer A the polynucleotide binding sites of Si is involved in the inter- (containing 5 mM Mg2+), ribosomes partially dissociate and action with 30S ribosomal subunits (presumably with exposed sediment as distinct 70S, 50S, and 30S peaks; the distribution portions of the 16S rRNA) and which sites might remain available for binding of mRNA or other nucleic acid compo- nents. Furthermore, if competition between polynucleotides Table 2. Protein Si-ribosome binding constants determined by and 30S particles for Si is observed, the relative binding af- competition measurements* finities can be used to obtain an independent measure of Competitors for K3OS-S1, given the known association constants of SI for poly- Buffers 3HS1 bindingt Measured K nucleotides (5, 6). A (3) 30S vs. poly(rC) K30S-SI = 4.8 (+1.0) X 108 M- Fig. 2 shows sucrose gradients of [3H]S1 mixed with 30S 1 (3) 30S vs. poly(rC) subunits (depleted of Si) and with either denatured DNA or or poly(rA) K30S-Sl = 3.4 (±0.8) X 108 M-1 poly(rC). Again the positions of the various bands were defined A 70S = (but not perturbed) by the tracer amounts of Si used. Essentially (2) 30S vs. K3oS/K70S 1.2 (±0.3) all of the [3H]S1 protein was initially bound at the concentra- * Buffers are defined in Materials and Methods; all experiments were tions of subunits and polynucleotides used, and the distribution run at 18°C. 30S subunits had previously been depleted of endog- of [3H]S1 between the rapidly sedimenting peak due to 30S enous S1 and then reactivated (see text). All competition experi- ments were carried out at concentrations of 30S particles of ; 1 M. subunits and the more slowly sedimenting peak (10-15 S) due The number of independent determinations made of each binding to denatured DNA or poly(rC), reflects the competitive affinity constant are indicated in parentheses with the Buffer. K values are of Si for the two components. The results clearly show that shown ± SD. poly(rC) competes with 30S subunits for Si binding but dena- t Based on an intrinsic affinity of site II for poly(rC) of 1.0 X 106 M-l tured DNA does not. Similarly, competition occurs with poly- and for poly(rA) of -3.6 X 105 M-l (6). Because of the low concen- tration of Si protein relative to potential poly(rC) binding sites for (rA) and not with poly(dA) (not shown). Therefore, we conclude S1, the cooperativity of binding of S1 to poly(rC) can be ignored in that SI site I binding (RNA-DNA nonspecific) does not con- these experiments. In the calculation we assume one "strong" site tribute to the Si-30S interaction, and thus this site may be for SI binding on both the active and the inactive subunit (see available to bind other nucleic acids from solution without af- text). Downloaded by guest on September 24, 2021 Biochemistry: Draper and von Hippel Proc. Nati. Acad. Sci. USA 76 (1979) 1043 of [3H]S1 among these peaks provides a measure of the relative affinity of Si for these three types of particles. We found (data not shown) that the 50S subunits did not bind SI detectably under the conditions of the experiment, indicating that the 50S subunit carries no Si binding site with affinity greater than ;107 M-'. The binding constants of 30S and 70S particles for Si protein were the same within experimental error (Table 2), 15. suggesting that 70S particle formation does not appreciably E perturb Si binding to the 30S subunits. Similar results have been obtained by Laughrea and Moore (10). 4.'6 Interaction of SI Protein with "Inactive" 30S Ribosomal Particles. In low-salt buffers, 30S ribosomal subunits undergo g1o II a reversible conformational change to a form that is termed "inactive," in that these subunits are not efficient in facilitating protein synthesis (9). Some of the manifestations of this ac- C., _ , tive-inactive conformational transition include changes in the exposure of some sulfhydryl groups of proteins (including SI) 5. ~~~~~~~~b associated with the 30S particle (15) and increased exposure of single-stranded regions of 16S rRNA (16). Laughrea and Moore have published data that can be interpreted in terms of one "strong" SI binding site per active 30S particle, with several additional "weak" Si binding sites being exposed on the inactive 0 - 30S subunit (10, 13) (also see Discussion). 10 15 20 24 As pointed out above, the band sedimentation method gen- Fraction erally can detect and quantitate only the strongest class of FIG. 3. Dependence of the binding affinity of Si protein for binding sites. We found that the affinity of SI for inactive 30S "inactive" 30S ribosomal subunits on Si protein concentration. Sl-depleted 30S subunits (at a concentration of 30 nM) were sedi- subunits is 5.6 X 108 M-', compared to t2 X 108 M-1 for the mented with [3H]Si protein (total concentration <0.i nM) in buffer strong site on active subunits (see Table 1). This small increase I; to one sample (0-0) was added 17 nM unlabeled Si protein. The in apparent affinity might reflect merely the difference in two samples were sedimented simultaneously. Mg2+ concentration required to maintain these different con- formations; alternatively, a real (small) difference in the ribo- subunits with increased S1 concentration, in keeping with some somal binding site for SI may be responsible for this affinity contiguous (and cooperative) binding of S1 to inactive 30S change. Effective competition for S1 protein by poly(rA), but particles. Identical experiments with active subunits showed not by poly(dA), again implicates site II (and not site I) as the no detectable dependence of K3OS-Sl on S1 concentration. major source of interaction free energy in binding S1 to inactive 30S ribosomal subunits. We conclude, in accord with Laughrea DISCUSSION and Moore, that the major conformational changes of the 30S The experiments reported here, together with previous work subunit involved in the active to inactive transition have little on the polynucleotide binding properties of S1, allow us to draw or no effect on the strongest binding interaction of this particle several conclusions about the way S1 binds to ribosomes. with Si protein. (i) The strongest binding affinity of Si protein for active and Because site II of S1 protein can bind cooperatively to for inactive 30S subunits (and for 70S ribosomes), measured by polypyridines, and more than one S1 molecule can bind to in- band sedimentation and competition methods, isv2 X 108 M-'. active 30S subunits (10, 13) (see Discussion), it is possible that Approximately one S1 protein molecule per ribosome or ribo- two or more S1 proteins might bind cooperatively to a stretch somal particle is bound with this affinity. Additionally, S1 ti- of single-stranded 16S rRNA in these particles [about 20 bases trations of active 30S subunits labeled with a fluorescent are required for cooperative site II binding (6)]. Neither the ethenoadenosine derivative at the 3' terminus of the 16S rRNA band sedimentation nor the competition experiments reported confirm this binding constant and also suggest a (strong) binding here would detect such cooperativity because the excess of stoichiometry of approximately one S1 per 30S subunit (B. potential binding sites over S1 protein (>100-fold) would Hindman, unpublished observations, this laboratory). swamp the modest cooperativity factor (;30). However, if the (ii) The competition experiments demonstrate that site I of concentration of S1 were raised to a level in excess of -0. 1 S1 S1 is not utilized in the binding of S1 to ribosomes and is thus per 30S particle, an increase in the apparent binding constant potentially free to interact with mRNA. Others have shown that would be seen if cooperative binding were involved. the sulfhydryl reagent N-ethylmaleimide does not affect the Fig. 3 shows the results of a pair of sedimentation runs on capacity of S1 to bind to ribosomes but does inactivate the [3H]S1 protein mixed with inactive 30S particles. These ex- ability of S1 to stimulate mRNA binding to ribosomes (13, 17) periments were performed identically, except that 0.57 copy (see also ref. 18). The ability of site II to inhibit protein synthesis of unlabeled S1 per 30S subunit was added to one sample. The at high Sl-to-ribosome ratios is unaffected by N-ethylmalei- run with a higher concentration of S1 showed a greater pro- mide (17). These findings are consistent with a possible inter- portion of protein migrating with the 30S peak and hence a action of site I (but not site II) with mRNA during initiation. higher apparent binding affinity. This change in sedimentation (iii) Site II either must be buried when S1 binds to ribosomes, pattern was quite reproducible. Sedimentation of 30S particles so that interaction with RNA and ribosomes simultaneously is at the concentrations of Fig. 3, resulting in only a small portion impossible, or (the simpler hypothesis) it must interact directly of the S1 protein remaining with the SOS peak by the end of the with the 16S rRNA. S1 is not easily removed from 30S subunits; run, makes the sedimentation pattern sensitive to very small even at very high salt concentrations, considerable dilution of changes in K30S-S1 (12). These results can be used to estimate the system is required (19). These results are consistent with the a 2- to 3-fold increase in the apparent K30S S1 for inactive known magnitude and lack of salt-sensitivity of the binding Downloaded by guest on September 24, 2021 1044 Biochemistry: Draper and von Hippel Proc. Nati. Acad. Sci. USA 76 (1979) constant of site II from RNA (6). However, although a major with 51), the ratio of S1 to 30S in the 30S peak after sedimen- part of the free energy of binding of Si to the 30S ribosomal tation will be significantly lower than the true binding stoi- particle may be due to thebinding of site II of the protein to a chiometry, unless the binding constant is exceptionally large. particular region of the 16S ribosomal RNA, the free energy of We calculate the maximum observable binding stoichiometry this interaction [--8.0 kcal (1 kcal = 4.18 kJ)/mol; see ref. 6] for a site with K - 2 X 108 M-1, under the sedimentation is not sufficient to account for the total free energy of S1-30S conditions of Laughrea and Moore (13), to be t0.8 S1 per 30S binding ('--10.9 kcal/mol). Therefore, additional interactions particle, close to the value of 0.9 (+0.1) observed by these must be involved. workers for active 30S subunits. Recently, Laughrea and Moore (20) demonstrated a sub- We are grateful to Mrs. Ruth Draper for excellent technical assis- stantial decrease in Si binding affinity for 30S particles re- tance in many phases of these studies and to our laboratory colleagues constituted without S9 protein. Omission of S9 and several other and Dr. Peter Moore for helpful comments on an earlier draft of this proteins from a 30S particle reconstitution mixture results in manuscript. This research was supported in part by U.S. Public Health a decrease in the (favorable) S1 binding free energy of -1.8 Service Research Grant GM-15792 and by a Predoctoral Traineeship kcal/mol (calculated from the relative affinity of S1 for com- (to D.E.D.) from U.S. Public Health Service Training Grant GM- plete 30S and "core" 30S particles reported in ref. 20). Thus, 00444. the free energy of binding of S1 to 30S subunits can be largely 1. van Duin, J. & van Knippenberg, P. H. (1974) J. Mol. Biol. 84, accounted for by the sum of the (nonspecific) affinity of site II 185-195. for polyribonucleotides and a specific interaction between S1 2. Chang, C. & Craven, G. R. (1977) J. Mol. Biol. 117,401-418. and S9. Based on this calculation, it seems unnecessary to pos- 3. Tal, M., Aviram, M., Kanarek, A. & Weiss, A. (1972) Biochim. Biophys. Acta 281, 381-392. tulate any rRNA sequence or structural recognition capacity 4. Draper, D. E., Pratt, C. W. & von Hippel, P. H. (1977) Proc. Nati. for site II; the binding of S1 protein to a unique site on the 16S Acad. Sci. USA 74,4786-4790. ribosomal RNA is ensured by the small additional free energy 5. Draper, D. E. & von Hippel, P. H. (1978) J. Mol. Biol. 122, of interaction of Si with other specific 30S proteins (20). We 321-338. note that colicin-treated 30S subunits (which have lost a 50- 6. Draper, D. E. & von Hippel, P. H. (1978) J. Mol. Biol. 122, nucleotide sequence at the 3' end of the 16S rRNA of the par- 339-359. ticle) still bind Si protein strongly (13). This suggests that some 7. Dahlberg, A. E. & Dahlberg, J. E. (1975) Proc. Natl. Acad. Sci. portion of the 16S rRNA other than the 3' terminus is involved USA 72,2940-2944. 8. van Dieijen, G., van Knippenberg, P. H. & van Duin, J. (1976) in the tight Si binding observed to active 30S particles. Eur. J. Biochem. 64,511-518. The values of K30s8S1 calculated here are 1 to 2 orders of 9. Zamir, A., Miskin, R. & Elson, D. (1971) J. Mol. Biol. 60,347- magnitude larger than those reported by Laughrea and Moore 364. (10, 13), who used band sedimentation data obtained with 30S 10. Laughrea, M. & Moore, P. B. (1977) J. Mol. Biol. 112, 399- subunits in the presence of varying excesses of Si protein to 421. estimate binding parameters. This discrepancy is due to the 11. Hill, W. E., Rossetti, G. P., van Holde, K. E. (1969) J. Mol. Biol. (implicit) assumption by these authors that no reequilibration 44,263-277. to ribosomes or ribosomal particles 12. Draper, D. E. & von Hippel, P. H. (1979) Biochemistry, in of the S1 protein bound press. ci ,i occurs during sedimentation, whereas we assume that ree- 13. Laughrea, M. & Moore, P. B. (1978) J. Mol. Biol. 121, 411- quilibration is complete at each level of the band sedimentation 430. experiment (see discussion and justification of this point in ref. 14. Harrington, W. F. & Kegles, G. (1973) Methods Enzymol. 27, 12 and footnote t). Such continual reequilibration leads to a 306-345. substantial loss of S1 protein from the complex; we have shown 15. Ginzburg, I. & Zamir, A. (1975) J. Mol. Biol. 93,465-476. that binding constants calculated without taking this loss of 16. Hogan, J. J. & Noller, H. F. (1978) Biochemistry 17,587-593. protein into account are usually at least an order of magnitude 17. Kolb, A., Hermoso, J. M., Thomas, J. 0. & Szer, W. (1977) Proc. Moore Nati. Acad. Sci. USA 74,2379-2383. too low (12). Recalculation of the data of Laughrea and 18. Thomas, J. O., Kolb, A. & Szer, W. (1978) J. Mol. Biol. 123, (10, 13) on an equilibrium basis bring their results and ours into 163-176. good quantitative accord. 19. Steitz, J. A., Wahba, A. J., Laughrea, M. & Moore, P. B. (1977) The loss of S1 protein from the sedimenting band due to Nucleic Acids Res. 4, 1-15. reequilibration also means that no matter how large the initial 20. Laughrea, M. & Moore, P. B. (1978) J. Mol. Biol. 122, 109- excess of Si over 30S subunits (i.e., all 30S subunits saturated 112. Downloaded by guest on September 24, 2021