Biochimie (1991) 73, 1245-1247

© Soci6t6 fran¢aise de biochimie et biologie mol6culaire / Elsevier, Paris

1245

The initiation codon AUG binds at a hydrophobic site on yeast 40S ribosomal subunits as revealed by fluorescence studies with bis (1,8-anilinonaphthalenesulfonate) JC Lee, LCC Yeh, PM Horowitz Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX, 78284-7760, USA

(Received 4 April 1991; accepted 24 May 1991)

Summary m Binding, studies of yeast 40S ribosome with bis (I ,8-anilinonaphthalenesulfonate) (bis-ANS) revealed the binding of 3-4 molecules of bis-ANS per ribosome with a dissociation constant (Kd) of 1.45 l.tM. Binding of AUG to the 40S subunits resulted in a concentration-dependent decrease in the bis-ANS fluorescence without displacing all of the bound bis-ANS from the ribosomes. The residual bis-ANS fluorescence at saturation with AUG corresponds to about 3 molecules of bis-ANS per ribosome. Thus AUG displaces one of the bound bis-ANS molecules. The data suggest that AUG binds at a hydrophobie site on the yeast 40S subunit. yeast / ribosome / mRNA binding / fluorescence / protein hydrophobic surfaces

Introduction

Materials a n d m e t h o d s

R i b o s o m e - m R N A interaction is an important early step in the process o f protein synthesis [1, 2]. For eukaryotes, components constituting the m R N A binding sites on rat liver and rabbit reticulocyte ribosomes have been identified by several experimental approaches. By chemical cross-linking, ribosomal components that could be attached to the m R N A in the ribosome-mRNA complex have been identified [3-7]. Some of these components are protected from chemical modifications in the presence of synthetic m R N A and are implicated in m R N A binding [8-10]. However, the chemical nature of the m R N A binding sites has not been elucidated. Information on the identity o f the yeast ribosomal components composing the m R N A binding site and the chemical nature of the site are not available. In the present study, the fluorescent dye bis (1,8anilinonaphthalenesu!fonate) (bis-ANS) was used to determine the effect o f binding of the initiation codon triplet A U G on the accessibility of hydrophobic surfaces on the yeast 40S ribosome subunit. The dye has been shown to be a sensitive reporter of solventaccessible apolar regions o f proteins and undergoes fluorescence enhancement upon binding to hydrophobic regions of proteins [ 11, 12].

Materials

Bis (l,8-anilinonaphthalenesulfonate) and the trinucleotide AUG were purchased from Molecular Probes, Inc (Junction City, OR) and Sigma Chemical Co (St Louis, MO), respectively. All other chemicals were reagent grade. Yeast 40S ribosome subunits were isolated from S cerevisiae (RNase 3A-strain) and purified by zonal centrifugation in a glycerol gradient as previously described [ 13]. Fluorescence measurements

Fluorescence intensities and spectra were measured at 25°C on a SLM/Aminco SPF-500C spectrofluorometer. The excitation wavelength for bis-ANS was 380 rim. Fluorescence cuvettes held 1 ml of sample and had a pathlength of 1 cm. A sample containing unmodified ribosomes was used as a blank for all fluorescence measurements. All determinations were made in triplicate. Temperature was controlled at 250C by circulating water through the cuvette holder. The concentration of bisANS was determined spectrophotometrically using an extinction coefficient of 21 120 m-i cm-l. To determine the number of bis-ANS bound at infinite concentration of AUG, the fluorescence intensities of different concentrations of 40S subunit in the presence of a fixed concentration of the dye (6 ~M) and different concentrations of AUG were measured. The reciprocals of the corrected fluorescence intensities (FI) vs those of the AUG concentrations were plotted. The plot was used to determine the fluorescence

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JC Lee et al

intensity at infinite AUG for a given amount (A260) of 40S subunit by extrapolatibn. Each fluorescence intensity value was normalized to l A2~ of 40S subunit and compared to that in the absence of AUG. The fractional decrease in the fluorescence intensity at infinite AUG at each ribosomal subunit concentration was used to establish the fractional decrease in the number of bound dye.

Results and discussion

Anilinonaphthalenesulfonates (ANS), such as 1,8ANS, 2,8-ANS, and bis-ANS, have been used extensively as sensitive reporters of solvent-accessible apolar regions on the surface of proteins [ 11, 12]. The present study showed the presence of apolar regions on the yeast 40S ribosomal subunit. Figure 1 (insert) 0.150

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B Fig 1. Scatchard plot for the binding of bis-ANS to yeast 40S ribosome subunits. The concentration of bis-ANS was determined spectrophotometrically using an extinction coefficient of 21 120 m-! cm-I. The fluorescence intensity of the dye bound to 40S ribosomal subunits was determined by the addition of increasing amounts of subunit to 22 ~tM bis-ANS and corrected for the fluorescence intensity contributed by the blank. The reciprocals of corrected fluorescence intensities were plotted as a function of the reciprocals of ribosome concentrations and extrapolated to infinite ribosome concentration. The concentration of the yeast 40S subunit was determined assuming a molecular weight of 1.8 x 106 with an absorption coefficient of 1.0 at 260 nm for a concentration of 66 pg/ml. The value of the intercept was used as the fluorescence of totally bound bis-ANS. The concentration of bis-ANS bound to 40S subunit was determined by titrating a fixed amount of 40S subunit with increasing concentrations of bis-ANS. The amounts (in pM) of bound (B) and free (F) bis-ANS were determined using the intercept of the double reciprocal plot. Linear regression fits were calculated using the least-squares method. Data were analyzed by the method of Scatchard [14]. (Insert) Fluorescence emission spectra of bis-ANS bound to yeast 40S ribosome subunit (A) and to AUG (B). The spectra of 10 pM bis-ANS in standard buffer containing 10 mM Hepes, pH 7.8, 0.1 mM EDTA, 10 mM MgCI2, 50 mM KCI, and 10% glycerol are also shown (C).

depicts the fluorescence emission spectra of yeast 40S subunits in the presence of bis-ANS. The level of bisANS fluorescence in the buffer was insignificant. Upon binding of bis-ANS to the 40S subunit, a pronounced enhancement in the fluorescence intensity of the fluorescent probe was detected. The fluorescence intensity of bis-ANS was enhanced with increasing concentration of 40S subunit, indicating binding of increasing numbers of bis-ANS molecules to the ribosome. Figure 1 also shows a Scatchard plot [14] for bis-ANS binding to free 40S subunits. Least squares linear extrapolations from the data gave the average number of binding sites as 3--4 per 40S subunit. Whether aii si,es gave similar quantum yields could not be ascert: ]ned at present. From the reciprocal of the slope of the line, a dissociation constant (Kd) of 1.45 ~tM was derived. That the slope of the titration curve was reasonably linear without significant inflections suggests that the different binding sites are comparable in their affinity for bis-ANS. The present results are the first to show that the yeast small ribosome subunits contain exposed hydrophobic sites. The present data are reminiscent of the previous studies of Pochon and Ekert [10] showing binding of bis-ANS to E coil ribosomes. These authors showed that, at 10 mM magnesium, E coli 70S, 50S and 30S bind 3, 2, and 1 molecules of bisANS, respectively [15]. Thus, the yeast 40S subunit appears to bind a larger number of bis-ANS molecules than the E coli ribosome. Effects of AUG binding to 40S subunits on the binding characteristics of the bis-ANS were assessed. Addition of AUG to the reaction mixture containing a fixed concentration of 40S subunit resulted in a reduction in the fluorescence intensity (fig 2A). The reduction was dependent on the AUG concentration. Analysis of a reaction mixture containing 40S subunit and radioactive AUG on glycerol gradients revealed that the binary complex was formed under the present experimental conditions (data not shown). A stoiehiometry of approximately 0.8 molecule of AUG per 40S ribosome was determined. The observed changes in the fluorescence intensity resulting from AUG binding reflect either an average change in the quantum yield of the binding sites due to changes in the environment of the sites or a displacement of bis-ANS molecules from its binding sites. To distinguish these two possibilities, a titration study was carded out. The results shown in figure 2B demonstrate that binding of AUG to the 40S particle results in a concentration-dependent decrease in the bis-ANS fluorescence intensity. Extrapolation of the data to infinite AUG concentrations reveals that AUG binding does not lead to complete displacement of bound bis-ANS. The data further disclose an average of 2.8 molecules of bis-ANS are bound per 40S particle at infinite AUG.

Fluorescent studies of oligonucleotide binding

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Acknowledgments Supported by NIH (GM 35851) to JCL and by Welch (AQ723) to PMH. We also thank M Chrisman for his technical assistance in the preparation of yeast ribosomes.

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1 Kozak M (1983) Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbial Rev 47, 1-45 2 Pain VM (1986) Initiation of protein synthesis in mammalian cells. Biochem J 235, 625-637 3 Westermann P, Benndorf R, Lutsch G, Bielka H, Nygard O (1986) Arrangement of eukaryotic initiation factor 3 and mRNA within preinitiation complexes. In: Structure Function and Genetics of Ribosomes

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II^U0 Fig 2. A. Effects of AUG binding on the bis-ANS fluorescence were determined by measurement of the fluorescence intensity of 1 Az60 of yeast 40S subunit in the presence of 22 gM bis-ANS and increasing concentrations of the oligonucleotide. The small levels of fluorescence of the oligonucleotide in the presence of bis-ANS were subtracted. The spectra were corrected for the background of unlabeled 40S subunit alone. B. The fluorescence intensities of varying concentrations (0.2-1.0 A260) of ribosome were titrated with different amounts of AUG in the presence of a fixed concentration of bis-ANS at 6 l.tM. The reciprocals of the corrected fluorescence intensities (FI) were plotted as a function of the reciprocals of the AUG concentrations. Linear regression fits were calculated using the least-squares method.

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In conclusion, the present results have demonstrated the binding of 3-4 molecules of bis-ANS per 40S subunit and that AUG binding displaces one bisANS from the 40S subunit. It is tempting to speculate that hydrophobic interactions are involved in stabilizing the binding of the initiation codon AUG with the ribosome.

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(Hardesty B, Kramer G, eds) Springer-Verlag, NY, 642-657 Stahl J, Kobets ND (1981) Affinity labeling of proteins at the mRNA binding site of rat liver ribosomes by an analog of octauridylate containing an alkylating group attached to the Y-end. FEBS Lett 123, 269-272 Takahashi Y, Ogata K (1981) Ribosomal protein crosslinked to natural mRNA by UV irradiation of rat liver polysomes. J Biochem 90, 1549-1552 Terao K, Ogata K (1979) Proteins of the small subunits of rat liver ribosomes that interact with poly U. I. Effects of preincubation of poly U with 40S subunits. J Biochem 86, 597-603 Terao K, Ogata K (1979) Proteins of the small subunits of rat liver ribosomes that interact with poly U. II. Crosslinks between poly U and ribosomal proteins in 40S suhunits induced by UV irradiation. J Biochem 86, 605-617 Westermann P, Heumann W, Bielka H (1975) Changes in the react;vity of proteins of rat liver ribosomes against [14C]iodoacetamide depending on their organization in ribosomal subparticles, 80S ribosomes, and on attachment of poly U. Chem Biol Interaction 10, 429-439 Gross B, Westermann P (1976) Studies on proteins of animal ribosomes. XXIV. Localization of proteins in ribosomal subunits of rat liver studied by chemical substitution with P-nitrophenyl acetate and methyl acetimidate. Chem Biol Interaction 15,309-317 Bielka H, Stahl J (1978) Structure and function of eukaryotic ribosomes. In: MTP Inter Rev Biochem (Amstein HRV, ed) University Park Press, Baltimore 18, 79-168 Rosen CG, Weber G (1969) Dimer formation from 1aniline 8-naphthalenesulfonate catalyzed by BSA. A new fluorescent molecule with exceptional binding properties. Biochemistry 8, 3915-3920 Brand L, Gohlke JR (1972) Fluorescence probes for structure. Annu Rev Biochem 41,843-868 Lee JC, Henry B, Yeh YC (1983) Binding of proteins from the large ribosomal subunits to 5.8S rRNA of S cerevisiae. J Biol Chem 258, 854-858 Scatchard G (1949) The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51, 660672 Pochon F, Ekert B (1973) Fluorescent labels on ribosomes. Action of the carcinogen 7-bromomethylbenz(a)anthracene. E u r J Biochem 36, 311-316