Interaction of Escherichia Coli Ribosomal Protein S1 with Ribosomes
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Proc. Nati. Acad. Sci. USA Vol. 76, No. 3, pp. 1040-1044, March 1979 Biochemistry Interaction of Escherichia coli ribosomal protein S1 with ribosomes (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-ribosome 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 proteins 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 ribosomal protein 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 translation 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.