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Chemical inactivation of Escherichia coli 30 s ribosomes with maleic anhydride: Identification of the proteins involved in polyuridylic acid binding
J. Mol. Bid. (1977) 115, 389-402
Chemical Inactivation of Escherichia coli 30 S Ribosomes with Maleic Anhydride : Identification of the Proteins Involved in Polyuridylic Acid Binding WCHAEL
CAXTRELL
AND
GARY R. CRAVEN
Laboratory of Molecular Biology and Department of Genetics University of Wisconsin Madison, Wise. 53706, U.S.A. (Received 7 February 1977) Modification of 30 S ribosomal subunits by the protein-modifying reagent maleic anhydride was found to inaotivate the particles for polyuridylic aoid binding. Reconstitution of 30 S ribosomes using 16 S RNA, maleylated total 30 S protein, and purified, unmodified proteins demonstrated that S4, Sll, S12, 813 and S18 are involved in poly(U) binding. Modified 30 S subunits contain all the ribosomal proteins and show normal sedimentation characteristics, indicating that the inaotivation is not simply due to the gross alteration of the particles. Correlation of these results with those of other workers is discussed.
1. Introduction In recent years a concerted effort has been directed toward understanding the functional roles of the ribosomal components. A number of approaches have been devised to determine the relative importance of the ribosomal proteins in specific functions. In light of the immense complexity of the Escherichia wli ribosome, it has become evident that definitive identification of the proteins involved in any function can only be made after similar results are obtained by a number of approaches. Thus several independent experimental approaches have been utilized to determine the components involved in messenger RNA and transfer RNA binding to the 30 S subunit. One of the early methods devised was the use of reconstitution experiments to study the effect on various activities of altering the component composition of the 30 S subunit (Nomura et al., 1969; Ozaki et al., 1969; Van Duin $ Kurland, 1970; Held et al., 1974). Another method employed protection studies to identify the components protected against chemical or enzymatic attack by the attachment of mRNA and tRNA (Noller et al., 1971; Rummel & Noller, 1973). A number of studies have been conducted to determine the ability of antibodies or antibody fragments specific for individual ribosomal proteins to block binding sites (Lelong et al., 1974; Sttiffler, 1974). Afllnity labels have been used to determine which proteins are physically located in the active sites (Pellegrini et al., 1974; Fiser et al., 1975; Pongs et al., 1975). More recently, chemical inactivation of the 30 S ribosome followed by reconstitution experiments has allowed identification of the components functionally involved in specific binding activities (Fanning, 1971; Thomas et al., 1975; Shimizu & Craven, 1976). 389
390
M. CANTRELL
AND
G. R. CRAVEN
We report here the application of chemical modification to identify the proteins at the site of inactivation for poly(U) binding. Modification of 30 S subunits is performed with maleic anhydride, a chemical reagent which primarily modifies free amino and sulfhydryl groups (Butler et al., 1967J969). This modification results in complete loss of poly(U) binding activity. We describe experiments in which reconstitution of 30 S subunits using 16 S RNA, modified 30 S total protein, and selected unmodified proteins allows us to identify proteins 54, Sll, 512, 813 and 518 as being at the site of inactivation for poly(U) binding.
2. Materials and Methods (a) Preparation
of ribosomes
and puti$ed
ribosornal
proteins
30 S ribosomes were isolated from E. coli MREGOO and purified as described previously (Craven 8.z Gupta, 1970). Ribosomal proteins were extracted from the purified 30 S subunits with 67% (v/v) acetic acid and fractionated by column chromatography with phosphocellulose (Mannix-P,, high capacity) following the procedure described by Hardy et al. (1969). In some cases the proteins were further fractionated by chromatography on carboxymethyl-cellulose (Whatman CM52) using the same buffers and elution conditions as applied in the phosphocellulose column chromatography. Identification of the proteins was established by a combination of chromatographic properties, 1 -dimensional polyacrylapolyacrylamide gel mide gel electrophoresis (Hochkeppel et al., 1976), and 2dimensional electrophoresis (Schendel & Craven, 1976). Sll and S9 were identified by immunodiffusion (Kahan et al., 1974) with antibodies supplied by L. Kahan and co-workers (University of Wisconsin). 30 S ribosomes labeled with 3H in wiwo were prepared by growing E. co&i MREBOO in lysine assay medium (Difco) containing [3H]lysine (New England Nuclear). Isolation of the ribosomes was as described above. (b) Male&
anhydride
modi$cation
of 30 S subunits
30 S subunits were heat-reactivated by incubation at 42°C for 20 min in the activation acetate, 0.2 Mbuffer of Zamir et al. (1971) : 50 mrvr-Tris.HCl (pH 7.2), 20 mM-mctgneSiUm NH,Cl, 2 mM-dithiothreitol. The ribosomes were then extensively dialyzed against poly(U) binding buffer (10 mivr-Tricine (pH adjusted to 7.6 with KOH), 20 m&r-magnesium acetate, KC1 to bring K+ concentration to 50 IIIM). The ribosomes remained in a heatreactivated state following this dialysis as defined by tRNA binding activity at 4°C. Maleic anhydride modification was then performed ss follows. 30 S subunits at 50 A,,, units/ml in poly(U) binding buffer were preincubated 2 min at 28”C, and the reaction was initiated by the addition of a solution of maleic anhydride (Aldrich) dissolved in dioxane (Aldrich, spectrophotometric grade) to give a molar ratio of maleic anhydride to 30 S subunits of 200 unless otherwise indicated. The volume of dioxane was always equal to or less than 5% of the volume of 30 S, an amount shown to have no effect on 30 S structure and adtivity (results not shown). Incubation was for 10 min at 28°C followed by addition of an excess of dithiothreitol. Stock [14C]maleic anhydride (Amersham-Searle) had a spec. act. of 21 mCi/mmol. (c) In vitro
functional
assay8
[3H]poly(U) binding assays were performed by the method of Smolarsky t Tal (1970) with the following modifications. Nitrocellulose filters (Millipore Corp. ; HAWP) were treated with 0.5 M-KOH for 30 min at room temperature. Unless otherwise indicated, 0.1 in 0.45 or O-9 ml of poly(U) binding buffer plus or 0.2 AzGO units of 30 S were incubated 1 maa-dithiothreitol with an excess of [3H]poly(U) (Schwarz-Mann; 11.5 to 22 mCi/mmol for 5 min at O’C). Approx. 0.5 pg of [3H]poly(U) per O-1 A,,, unit of 30 S was usually used. Samples were then filtered as described by Smolarsky & Tal (1970), and the dried filters were counted in 10 ml of a scintillation fluid composed of 5 g PPO and 0.1 g POPOP/l
305 PROTEINS
INVOLVED
IN POLY(U)
BINDING
391
toluene. Due to the variable polymer length of the [3H]poly(U), it is not possible to compute the absolute percentage of ribosomes active for poly(U) binding. The variations from Tables 1 to 4 in cts/min of [3H]poly(U) bound per Aaeo umt of 30 S ribosome are due to 2 factors. First, a number of different batches of [3H]poly(U) were used which had a 2-fold range in specific activities. Second, differences in chain length of poly(U) over the course of this study resulted in different numbers of cts/min bound per ribosome. The activity in the poly(U) assay was determined to be directly proportional to the ribosome concentration within each set of data. The poly(U)-directed [14C]phenylalanyl-tRNA binding activity of the ribosomes was measured by the method of Nirenberg & Leder (1964). About O-5 A,,, unit of 30 S particles w&9 assayed in 1 ml of a binding buffer containing 0.1 M-Tris*HCl (pH 7*2), 20 mM-magnesium acetate, 50 mm-KCl, 1 mnil-dithiothreitol, 40 pgpoly(U), 2.81 Azso units of total E. coli B tRNA (Schwartz-Mann) charged with [14C]phenylalanine (Amershsm-Searle, spec. act. 500 mCi/mmol Phe). The samples were incubated 10 min at 37”C, then diluted with 3 ml of the cold binding buffer and applied to a nitrocellulose filter which was washed twice with 3 ml of buffer. Filters were counted in 10 ml of a scintillation fluid composed of 5 g PPO, 60 ml Bio-Solv BBS-3 (Beckman) and toluene to 1 1. of total 30X protein and RNA (d) Exkzction Total 30 S protein and 16 S RNA were extracted from control and maleic anhydridemodified 30 S subunits by a modification of the procedure of Spicer & Craven (manuscript in preparation). The procedure allows extraction of the protein and RNA without exposing them to acidic conditions, which would partially reverse the mdeio anhydride modification (Butler et al., 1969; Glazer, 1976). A suspension of 305 subunits WMI dialyzed against the extraction buffer of 6 M-Urea, 60 mM-Tris*HCl (pH 7*6), 40 mM-magneSiUm acetate, 0.66 M-KCl, 2 mM-dithiothreitol at 4°C. Samples were incubated 1.5 h at room temperature with intermittent shaking and layered over 2 ml of 5% (w/v) sucrose in the extraction buffer in Beckman 50 Ti tubes. Samples were then centrifuged 15.5 h at 34,700 revs/min. The purified protein (supernatant) was dialyzed at 4’C verau8 5 M-Urea in RB2 buffer (30 mM-Tris*HCl (pH 7.6), 20 mM-UX3gUeSiUm acetate, 0.5 M-KCl, 1 mM-dithiothreitol). Portions were frozen and stored at - 70°C. The purified RNA pellet was redissolved in and dialyzed versus 10 mM-Tris*HCl (pH 7.6) and stored at -70°C. (e) Reconstitution and isolaGon of subunits With a few modifications, the reconstitution system described by Held et cd. (1973) was used. Proteins in 5 M-ureeRB2 buffer were dialyzed into RB2 buffer just prior to their use. The 16 S RNA was dialyzed into 30 mm-Tris+HCl (pH 7.6), 20 mna-magnesium acetate, 1 mM-dithiothreitol. RNA was then incubated 10 min at 42°C and the protein was added to the RNA at an appropriate volume to give the salt concentration of reconstitution buffer (30 ma/r-Tris*HCl (pH 7*6), 20 mM-magneSiUm acetate, 0.33 M-KC& 1 mxl-dithiothreitol). The incubation was continued for 60 min at 42°C. Unless otherwise indicated, reconstitutions were performed at a concentration of 1.5 A,,, units RNA/ml and at a molar ratio of protein to RNA of 2.5: 1. The molar ratio of individual proteins and the concentration of RNA were determined &9 described previously (Hochkeppel et al., 1976). Particles reconstituted from both modified and unmodified components showed normal sedimentation characteristics. After cooling to 4”C, the reconstituted particles were isolated by precipitation at 0°C with 0.5 vol. absolute ethanol (Staehelin et al., 1969; Thomas et al., 1975). The particles were resuspended in 0.4 to 0.6 ml of reconstitution buffer, and any undissolved materials were removed by low-speed centrifugation. Prior to testing for activity, the particles were then heated for 20 min at 42°C.
(f) Sucrose gradient centrifugation and polyacrylamide gel electrophoresis Samples containing 2 A,,, units of ribosomal subunits were layered on 3*5-ml 5% to 20% linear sucrose gradients in poly(U) binding buffer, centrifuged for 1.5 h at 55,000 revs/min at 4°C (Beckman SW56 rotor), and analyzed by pumping directly through a l-mm flow-through cell in a Beckman DB-G spectrophotometer. 26
392
M. CANTRELL
AND
G. R. CRAVEN
Two-dimensional gel electrophoresis was carried out essentially as described previously (Schendel & Craven, 1976). A total of 15 to 30 A 280 units of 30 S ribosomes was dialyzed into 6 M-urea, 0.03 M-Tris (pH 7*6), 5 mM-dithiothreitol and treated with pancreatic ribonuclease prior to electrophoresis. The proteins were separated in the first dimension by disc gel electrophoresis as described by Howard & Traut (1973) for 1 h at 3 mA/tube, then 5 to 7 h at 6 mA/tube. The proteins were separated in the second dimension by slab gel electrophoresis as described by Martini & Gould (1971) with slight modifications. The 2-dimensional gel apparatus was identical to that of Mets & Bogorad (1974). The first dimension gels were dialyzed at 37°C against: (1) 1 M-phosphate (pH 7-O), 1.0% (w/v) sodium dodecyl sulfate (30 min) ; (2) 0.01 M-phosphate (pH 7*1), l.Oo/o sodium dodecyl sulfate (20 min) ; and (3) 0.01 M-phosphate (pH 7*1), 0.10% sodium dodecyl sulfate (25 min) in preparation for the second dimension. The second dimension gel solution was identical to that of Martini & Gould (197 1) except the amount of N,N-methylene bisacrylamide was doubled. After polymerizing the l-dimensional gels onto the second dimension slab, electrophoresis proceeded for 15 to 16 h at 60 V.
3. Results (a) Modijimtion
of 30 X subunits with muleic anhydride
Figure 1 shows that the reaction of 30 S subunits with maleic anhydride results in the loss of both poly(U) and Phe-tRNA binding activities. Concomitantly, there is modification of the 30 S subunit as measured by the uptake of [14C]maleic anhydride (Fig. 2). There are three possible explanations for this inactivation : (1) loss of activity is due to direct inactivation of the site for poly(U) binding; (2) modification of the subunit results in a loss of ribosomal proteins, thereby causing inactivation; (3) modification causes a conformational change in the subunit which leads to loss of activity.
Moles mleic anhydride added/3OS subunit
FIG. 1. The effect of meleic anhydride on poly(U) and Phe-tRNA binding activity. The 30 S subunits were modified as described in Materials and Methods with varying amounts of maleio anhydride. Numbers on the abscissa represent the ratio of maleic anhydride to subunit in the reaction mixture. The reaction was terminated with an excess of dithiothreitol and assayed and poly(U)-directed phenylalanyl-tRNA binding directly for poly(U) binding (-•--~--) (-o-o-). A total of 0.47 &so unit of 30 S was used per assay sample for the sH-labeled poly(U) binding assay, and 100% activity corresponded to 8320 cts/min of 3H radioactivity. A total of 0.49 A,,, unit of 30 S was used per assay sample for the 14C-lebeled poly(U)-directed phenylalanyl-tRNA binding assay, and 100% activity corresponded to 2232 ots/min of 14C radioactivity.
305
PROTEINS
INVOLVED
IN
POLY(U)
BINDING
393
Moles maleic anhydride addedl3OS submit
Fm. 2. Uptake of [14C]maleio anhydride into 30s subunits. Subunits were modified as described in Materials and Methods with varying amounts of [l*C]maleic anhydride at a spec. act. of 1.388 mCi/ mmol. The reaction was terminated with an excess of dithiothreitol, and portions of 0.93 A 280unit were diluted to 2 ml with the reaction buffer. The samples were subsequently filtered on Millipore filters, washed, and counted as described in Materials and Methods. The plateau level of 8100 cts/min bound corresponds to 40 mol of maleic anhydride bound to each mol of 30 5. Backgrounds for any 14C cts/min of maleic anhydride products bound to Millipore filters in the absence of 30 S were subtracted.
Although identification of the 30 S components involved in the inactivation would yield valuable information no matter what the mechanism of inactivation, we were interested in determining which of the three potential mechanisms might be involved in the inactivation. Therefore, we initiated a series of studies to determine if the protein composition and/or the conformation of the modified particles had been altered. (b) Determination of the protein composition of modi$ed subunits To determine if any of the 30 S proteins substantially dissociate from modified subunits, 30 S particles were modified by maleic anhydride and isolated by centrifugation through a layer of 5% (w/v) sucrose in poly(U) binding buffer. Isolated control particles and isolated maleylated particles were analyzed for protein content by twodimensional gel electrophoresis as shown in Figure 3. Although the mobilities of many of the proteins are altered after maleylation due to the addition of negatively charged maleic acid groups, there does not appear to be any substantial loss of any protein as a consequence of the chemical modification. No conclusion can be drawn concerning the presence of 821 as it usually fails to appear in the gel system we have employed. Quantitation of the two-dimensional gels was not possible due to the alterations in electrophoretic mobility. As a result, it was possible that there could have been some general loss of ribosomal protein. To determine if this was the case, in &o-labeled ([3H]lysine) 30 S ribosomes were modified with maleic anhydride and centrifuged through sucrose as described above. Supernatant fractions and the pellet were then analyzed to determine the amount of [3H]protein in each. For control 30 S subunits and modified 30 S subunits, respectively, it was found that only 1.4% and 1.7% of the [3H]proteins were present in the supernatant fractions. It therefore appears that there is no substantial change in the protein composition of modified subunits.
394
M. CANTRELL
AND
G. R.
CRAVEN
FIG. 3. Two-dimensional gel electrophoresis of 30 S ribosomal proteins from isolated control (a) and isolated modified (b) 30 S subunits. (aI) Acidic protein, (aa) basic protein from control 30 S subunits. (b,) Acidic protein, (b,) basic protein from modified 30 S subunits.
(c) Mod$cation
does not cawe any large conformational change
It was also possible that maleic anhydride modification induced a conformational change in the particle, which in turn produced a loss in poly(U) binding activity. To test this possibility, modified and unmodified 30 S subunits were analyzed on sucrose gradients and the sedimentation constants determined. Figure 4 shows that the particles have normal sedimentation constants both before and after modification, even up to a ratio of 450 mol of maleic anhydride added per mol of 30 S present. The data therefore indicate that there are at least no gross alterations in the 30 S conformation. We therefore tentatively suggest that chemical derivatization of the 30 S subunit under these conditions leads to a loss of 90% of the poly(U) binding activity by direct
30s
PROTEINS
INVOLVED
IN
l?OLY(U)
BINDING
395
I Top -
Bottom Fractions
Fra. 4. Sedimentation of control 30 S subunits (a) and modified 30 S subunits (b). Modification was carried out as described in Materials and Methods at a ratio of 450 mol of maleic anhydride added per mol of 30 S. Samples containing 2 A,,,, units of 30 S in a volume of 46 ~1 were layered on sucrose gradients made up in the poly(U) binding buffer and centrifuged aa described in Materials and Methods.
modification of the components involved in that binding. This suggestion is further supported by the finding that modification of poly(U)-30 S complexes with maleic anhydride results in no loss of bound poly(U). (d) The protein component is modi$ed by maleic anhydride To determine whether maleic anhydride modifies the RNA or protein moieties of the 30 S subunit, a suspension of 30 S subunits was treated as described in Materials and Methods with a ratio of 228 mol of [14C]ma 1eic anhydride per mol of ribosomes in the reaction mixture. The protein and RNA were subsequently separated (see Materials and Methods), and each was dialyzed extensively versus its post-centrifugation extraction buffer and analyzed for [14C]maleic anhydride incorporation. Under these conditions, greater than 99% of the bound maleic anhydride remained associated with the protein fraction while less than 1% remained associated with the RNA fraction. We conclude that most, ifnot all, of the modification takes place on the protein component of the 30 S subunit. In support of this, two-dimensional gel electrophoresis of proteins extracted from maleic anhydride-inactivated ribosomes indicates that many of the proteins have altered electrophoretic mobilities due to modification (compare Fig. 3(a) and (b)). (e) Determination of the 30 S protein as the site of inactivation Heterologous reconstitution experiments were performed to determine whether the site of maleic anhydride inactivation for poly(U) binding was in the protein or the
396
M. CANTRELL
AND
G. R.
CRAVEN
RNA moiety of the 30 S subunit. The 16 S RNA and 30 S total protein were isolated from control and modified subunits. Reconstitutions and isolations were then performed with the combinations shown in Table 1, followed by measurement of the poly(U) binding capacity of each reconstituted particle. The site of inactivation is seen to be completely localized in the protein moiety. TABLE
Ident@c&on
of the protein moiety as the site of inactivation
source of RNA
c M c M
1
30s 305 30s 305
source of protein
c c M M
30s 30s 305 30s
Control activity after reconstitution (%I 100 98 19 17
RNA and total protein were extracted from unmodified (C 305) and modifled 305 subunits (M 305). Reconstitutions were then performed on all pairwise combinations with 2.6 molar equivalents of total protein per molar equivalent of RNA added, and the particles were isolated and assayed for poly(U) binding as described in Materials and Methods. The 100% activity represents the activity of particles reconstituted from unmodified protein and RNA. Its average value wss 8360 cts/min per 0.1 Ass,, unit of 305.
(f) Identi$cution
of the individual
proteins inuctivated by maleic anhydride
In order to identify the proteins at the site of inactivation, a series of reconstitution experiments were undertaken in which 16 S RNA and modified total protein were reconstituted in the presence of selected unmodified proteins. Preliminary experiments showed that the addition of more than one unmodified protein to the reconstitution mixtures of 16 S RNA and modified total protein was required to give an appreciable return of poly(U) binding activity. As a result, the 30 S proteins were divided into three groups to be studied for their ability to restore binding activity. Group 1 was composed of proteins Sl, S2, 55, 58, S9, Sll, S20 and S2I ; the composition of group 2 was proteins 53, 54, 512, 513, 514, 515, 518 and S19; and group 3 consisted of proteins S6, S7, SlO, S16 and 517. Table 2 presents averages of a number of experiments and shows that inclusion of either group 1 or group 2 proteins in the reconstitution mixture produced 30 S ribosomes with substantial poly(U) binding activity while the ribosomes obtained upon addition of group 3 proteins showed no poly(U) binding activity. Thus proteins S6, 57, SIO, S16 and 517 were eliminated as possible components of the site of maleic anhydride inactivation. It should be noted that the values for poly(U) binding given here are relative values for the activity of the particles and do not reflect the absolute percentage activity of the ribosome. The high percentage activity returned may either be a consequence of the variability commonly seen in reconstitution systems (Held et al., 1973) or a reflection of the fact that some of the ribosomal proteins have fractional stoichiometries (Craven et al., 1969 ; Hardy, 1975). All assays were determined to be strictly linear over the ranges used (see Materials and Methods). To determine which proteins within groups 1 and 2 were important for restoration
30s
PROTEINS
INVOLVED
IN
POLY(U)
BINDING
397
of poly(U) binding activity, a series of experiments was performed on each group separately in which the effect of the deletion of selected proteins from a group was ascertained (Table 3). Examination of Table 3 shows that the values obtained for percentage activity returned by different sets of proteins can be grouped into two distinct categories; those with activities close to lOO%, and those with activities much lower than 100%. TABLE
2
Restoration of poly( U) binahg activity by selectedprotein groups Proteins present during reconstitution MTP MTP MTP MTP MTP
+ + + +
control total protein group 1 proteins group 2 proteins group 3 proteins
Activity
returned (%) 0 100 83 172 0
Reconstitutions were performed on mixtures containing 1 molar equivalent of 16 S RNA plus 2.6 molar equivalents of modified tote1 protein and 2.6 molar equivalents of the indicated unmodified proteins. The reconstituted particles were then isolated and essayed for poly(U) binding activity. Group 1 contains proteins Sl, 52, S5, S8, SQ, Sll, S20 and S21; group 2 cont&s proteins 53, 54, 512, 513, 514, S16, 518 and SlQ; and group 3 contains proteins S6, 57, SlO, S10 cmd S17. The average activity for the 100% value was 3600 cts/min per 0.2 Aaso unit, end the average activity for the 0% value wss 310 cts/min per 0.2 Azao unit.
More specifically, there is a set of unmodified proteins whose deletion from the reconstitution results in activities within 22% of control, and there is a set whose deletion results in a reduction of activity by more than 54%. We suggest that the proteins directly required for poly(U) binding activity fall in the latter category. Table 3A thus shows that proteins Sl, 52, S5,58,520 and S21 can be eliminated as possible components at the site of maleio anhydride inactivation. Similarly, Table 3B shows that proteins S3,514,515 and S19 can be eliminated from group 2. As a result of the above studies, the proteins potentially involved in poly(U) binding were narrowed down to 512 and/or 513, 54 and/or 518, and Sll and/or S9. However, on the basis of those studies it could not be determined whether one or both of the proteins in each of these three pairs is important for activity. Final identification was thus done by reconstitutions using each protein in a highly purified form. Table 4 shows that deletion of any one of the implicated proteins except S9 results in a reduction in poly(U) binding activity. We therefore conclude that proteins 54, Sll, S12,513 and 518 are the major components of the site of inactivation for poly(U) binding. In addition, these proteins may directly be involved in the poly(U) binding site. We cannot, however, rule out the possibility that their modification may involve a small, undetectable conformational change in the subunit. 4. Discussion In a previous paper (Shimizu $ Craven, 1976) we reported the chemical inactivation of the 30 S ribosome for Phe-tRNA and fMet-tRNA binding by iodination. In that
398
M. CANTRELL
AND
G. R.
TABLE
CRAVEN
3
Restoration of activity by group 1 and group 2 proteins Proteins
present during reconstitution
Relative
A MTP MTP MTP MTP MTP MTP MTP MTP MTP
+ + + + + + + +
Sl, -, Sl, Sl, Sl, Sl, Sl, Sl,
52, SS, S8, S9, S2, SS, 58, S9, -, S5, S8, S9, 52, -, 58, S9, S2, S5, -, S9, 52, 55, S8, -, S2,55,58, S9, 52, S5, 58, S9,
Sll, Sll, Sll, Sll, Sll, -, Sll, Sll,
Expt 1 0 100
S20, S21 520, S21 S20,521 S20,521 520, S21 520,521 -, S21 520, -
96 127 40
B MTP MTP MTP MTP MTP MTP
+ + + + +
53, S4,512,513, 53,54,512,513, -, 54, S12,513, S3,--,S12,513, S3,S4, -, -,
S14, S15,518, -, -, S18, -, -, S18, -, -, -, -, -,S18,
S19 -
Expt 1 0 100 7.5
activity
returned
94 75 46 79 85
96 84
Average 0 100 94 113 95 101 43 88 85
Expt 2 0 100 95 78 36 36
Expt 3 0 100 93 80 48 54
Average 0 100 88 79 42 45
Expt 2 0 100 111
Expt 3 0 100
(%) Expt 4 0 100 76 113
Reconstitutions, isolations, and assays were performed as described in the legend to Table 2 and Materials and Methods. The average activity in A for the 100% value was 4630 cts/min per 0.2 in B for the A 260 unit and 310 cts/min per 0.2 A,,, unit for the 0% value. The average activity 100% value was 2930 cts/min per 0.2 A,,, unit and 460 cts/min per 0.2 A,,, unit for the 0% value. TABLE
Identi$cation of the proteins required Proteins present during reconstitution
4
for poly( U) binding activity Activity
returned 1%)
MTP MTP + control total protein MTP + S4, S9, Sll, 512,513,518 MTP + -, S9, Sll, 512, S13,518
0 100 118 82
(100) (69)
MTP MTP MTP MTP MTP
111 79 56 76 81
(94) (67) (47) (64) (69)
+ + + + +
54, -, Sll, 512, S13, S18 S4, S9, -, 512,513, S18 54, S9, Sll, -, 513, S18 S4, S9, Sll, 512, -, 518 Sr, S9, Sll, S12, 513, -
Reconstitutions, isolations, and assays were performed as described in the legend to Table 2 and Materials and Methods. The average activity for the sample containing 16 S RNA plus modified total protein and control total protein was 12,400 cts/min per 0.1 Azso unit. The average activity for the sample containing 16 S RNA plus modified total protein w&s 1700 ots/min per 0.1 A,,, unit. The numbers in parentheses are the rtotivity restored normalized to the activity of MTP plus the total complement of tested proteins.
30s
PROTEINS
INVOLVED
IN
POLY(U)
BINDING
399
study, reconstitution experiments analogous to those described here identified proteins 53,514 and 819 as being involved in fMet-tRNA binding and proteins Sl, S2, S3, S14 and S19 as being involved in Phe-tRNA binding. In this study we have extended that type of approach to identify the 30 S ribosomel proteins involved in poly(U) binding. Chemical modification with the protein-modifying reagent maleic enhydride has been shown to completely inactivate the 30 S ribosome for both poly(U) and PhetRNA binding. We have found by reconstitution that proteins 84, Sll, 512, 513 and 518 are critical proteins in returning this poly(U) binding activity. As Table 4 shows, it is probable that 512 is the most essential protein of the five proteins implicated. The observation that modification of amino groups by mctleic anhydride results in loss of poly(U) binding is not, by itself, strong evidence that amino groups are directly involved in poly(U) binding. However, Chang BECraven (manuscript in preparation) have surveyed a wide range of protein-modifying reagents and found that the only reagents inactivating the 30 S subunit for bacteriophage Qfi natural mRNA binding are either reagents which can cross-link the ribosome or which modify amino groups (with the exception of Rose Bengal, a histidine-derivatizing reagent). The amino acids modified in that study, which utilized over 25 protein-derivatizing reagents, include arginine, tyrosine, tryptophen, cysteine, lysine and the carboxyl groups of glutamate and asp&ate. There is thus a strong possibility that lysine amino groups are selectively involved in mRNA binding. It has been suggested that poly(U) binding to the ribosome is not specific (Nomura, 1970; Nomurrt & Held, 1974). Such suggestions are based partially on the fact that basic proteins alone can bind poly(U). However, there are a number of lines of evidence suggesting that the binding of poly(U) to the ribosome is specific. The earliest evidence for the specificity of poly(U) binding was the determination that poly(U) binds only to the 30 S subunit and not to the 50 S subunit (Takanami & Okamoto, 1963). It has been known for some time that aurintricarboxylic acid inhibits both natural mRNA and poly(U) binding to the ribosome (Grollman & Stewart, 1968). In addition Szer & Leffler (1974) have shown that poly(U) competes for MS2 RNA binding. We have found that prior binding of Q/3 RNA to unwashed 30 S ribosomes competes for the poly(U) binding capacity of those ribosomes (Cantrell & Craven, unpublished results). We would thus suggest that poly(U) and natural mRNA compete for the same site on the 30 S ribosome. Wagner & Gassen (1975) have shown that the oligonucleotide U-U-U-(nhr)2’U can direct Phe-tRNA binding while covalently bound to the 30 S ribosome. However, the most compelling indication of specificity of poly(U) binding comes from the identification of the proteins involved. If the binding were non-specific one might expect poor correlation with the results obtained using other mRNAs. However, this is not the case. The proteins found by our experiments to be involved in the poly(U) binding site are a distinct group of five proteins, which have been implicated in mRNA binding by a number of other approaches, using different mRNAs. One of these approaches has involved the use of chemically reactive mRNA analogs or affinity labels. Pongs e.tal. (1975) used this approach to identify the 30 S ribosomal proteins modified by attachment of a bromolated derivative of the AUG codon to 70 S and 30 S ribosomes. The proteins they have identified coincided exactly with our proteins, 54, Sll, 512,513 and X18, with the addition of S21. Similarly, Ltihrman et al. (1976) utilized an mRNA affinity label, G-U-U-iacn5U, and found attachment of Sl, S18 and 512. Fiser et al. (1975) implicated proteins Sl, 518 and 521 by the photoaffinity reaction of poly(4-thiouridylic acid) with 70 S ribosomes.
400
M. CANTRELL
AND
G. R. CRAVEN
Chemical modification studies have also produced much valuable information. Derivatization of 30 S ribosomes with Rose Bengal followed by reconstitution has implicated S2 and 53 to be involved in poly(U) and Phe-tRNA binding (Thomas et aE., 1975). However, there was only a partial inactivation (approx. 65%) of the poly(U) binding in this study. Chang & Craven (manuscript in preparation) have used the reagent 2-methoxy-5nitrotropone to identify Sl, 512, 513 and S21 as proteins essential for phage Q/I RNA binding. Reconstitution experiments by Nomum’s group have shown 811 and 512 to be very important for the ambiguity and fidelity of mRNA translation (Ozeki et al., 1969; Nomura et al., 1969). More recently they have also shown protein 512 as well as 16 8 RNA to be involved in determining the speci6city of initiation (Held et al., 1974). We have not found a requirement for unmodified Sl in our system. However, we would like to emphasize that this does not constitute a discrepancy with the many studies pointing to the importance of Sl in mRNA binding (Van Duin & Kurland, 1970; Noller et al., 1971; Piser et al., 1975). It may be that the necessary functions of Sl are merely not inactivated by maleic anhydride under our conditions. Structural studies of the 30 S ribosome suggest that the identified proteins may indeed be situated physically close to each other. Sommer & Traut (1974,1975) have produced cross-links of 54 and 513 and of 54 and S12. Recent work by that group (Sommer & Traut, 1976) has also identified 512 and 513 plus Sll and 513 as crosslinked products obtainable from the 30 S ribosome. In addition, immunoelectron microscopy (Lake & Kahan, 1975; Tischendorf et al., 1975) has shown that antigenic sites for 54, Sll, 512, 513 and 518 are in close proximity on the head region of the ribosome. The model produced by Stijffler and co-workers is shown in Figure 5 with selected antigenic sites for the proteins commonly implicated as being within the mRNA binding site. Due to the highly complex nature of the ribosome, independent information from a wide variety of experimental approaches is needed to unequivocally identify the
(a)
(b)
FIQ. 6. Model of the 30 S ribosomal subunit based on immunoelectron microscopy (Tischendorf et al., 1976). Two views are shown, giving selected antigenic sites for the ribosomel proteins commonly implicated in mRNA binding. The proteins identified in this study are circled by solid lines.
305
PROTEINS
INVOLVED
IN
POLY(U)
BINDING
401
components involved in mRNA binding. It is therefore encouraging to note the overwhelming agreement found by several independent techniques for a small group of ribosomal proteins. This combination of structural and functional information should ultimately give a much more coherent picture of the ribosome. The authors would like to offer special thanks to Dr T. Fanning whose preliminary studies provided the basis for this work. We are also indebted to Dr L. Kahan and D. Winkelmann for their help in immunochemical experiments and to MS C. Bloomer for providing purified ribosomal proteins. This work was supported by the Graduate School and the College of Agricultural and Life Sciences, University of Wisconsin, Madison and by research grant GM15422 from the National Institutes of Health. One of us (M. C.) was supported by training grant GM01874 from the National Institutes of Health. We dso acknowledge use of the University of Wisconsin Biochemistry Department pilot plant, directed by Dr J. Garver and supported by the United States Public Health Service grant Fr-00214.
Butler, Butler,
REFERENCES P. J. G., Harris, J. I., Hartley, B. S. & Leberman, R. (1967). Biochem. J. 103, 78. P. J. G., Harris, J. I., Hartley, B. S. & Leberman, R. (1969). Biochem. J. 112,
679-689. Craven, G. R. & Gupta, V. (1970). PTOC. Nat. Ad. Sci., U.S.A. 67, 1329-1336. Craven, G. R., Voynow, P., Hardy, S. J. S. & Kurland, C. G. (1969). Biochemistry, 8, 2906-2915. Fanning, T. (1971). Ph.D. Thesis, University of Wisconsin, Madison. Fiser, I., Scheit, K. H., Stoffler, G. & Kuechler, E. (1975). FEBS Letters, 56, 226-229. Glazer, A. N. (1976). In The Proteins (Neurath, H. & Hill, R. L., eds), vol. 2, pp. l-103, Academic Press, New York. Grollman, A. P. & Stewart, M. L. (1968). Proc. Nat. Acad. Sci., U.S.A. 61, 719-725. Hardy, S. J. S. (1975). Mol. Gen. Genet. 140, 252-274. Hardy, S. J. S., Kurland, C. G., Voynow, P. & Mora, G. (1969). Biochemistry, 8, 28972905.
Held, W. A., Mizushima, S. & Nomura, M. (1973). J. Biol. Chem. 218, 5720-5730. Held, W. A., Gette, W. R. & Nomura, M. (1974). Biochemistry, 13, 21152122. Hochkeppel, H., Spicer, E. & Craven, G. R. (1976). J. Mol. Biol. 101,155-170. Howard, G. A. & Traut, R. R. (1973). FEBS Letters, 29, 177-180. Kahan, L., Held. W. A. & Nomura, M. (1974). J. Mol. Biol. 88, 797-808. Lake, J. A. & Kahan, L. (1975). J. Mol. BioZ. 99, 631-644. Lelong, J. C., Gros, D., Gros, F., Bollen, A., Maschler, R. & Stoffler, G. (1974). Proc. Nat. Aeud. Sci.,
U.S.A.
71, 248-252.
Luhrman, R., Gassen, H. G. & Stoffler, G. (1976). Eur. J. B&hem. 66, l-9. Martini, 0. H. W. & Gould, H. H. (1971). J. Mol. BioZ. 62, 403-405. Mets, L. H. & Bogorad, L. (1974). Anal. B&hem. 57, 200-210. Nirenberg, M. & Leder, P. (1964). Science, 145, 1399-1407. Noller, H. F., Change, C., Thomas, G. & Aldridge, J. (1971). J. Mol. BioZ. 61, 669-679. Nomura, M. (1970). Bacterial. Rev. 34, 228-277. Nomura, M. & Held, W. (1974). In Ribosomes (Nomura, M., Tissieres, A. & Lengyel, P., eds), pp. 193-223, Cold Spring Harbor Laboratory Press, New York. Nomura, M., Mizushima, S., Ozaki, M., Traub, P. & Lowry, C. V. (1969). Cold Spring Harbor Symp. Quant. BioZ. 34, 49-61. Ozaki, M., Mizushima, S. & Nomura M. (1969). Nature (London), 222, 333-339. Pellegrini, M., Helen, O., Eilat, D. & Cantor, C. R. (1974). J. Mol. BioZ. 88, 809-829. Pongs, O., Stoffler, G. & Lanka, E. (1975). J. Mol. BioZ. 99, 301-315. Rummel, D. P. I% Noller, H. F. (1973). Nature New B&d. 245, 72-75. Schendel, P. L. & Craven, G. R. (1976). NucZ. Acids Res. 3, 3001-3014. Shimizu, M. & Craven, G. R. (1976). Eur. J. Biochem. 61, 307-315.
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AND
G. R. CRAVEN
Smolarsky, M. & Tal, M. (1970). Biochiwa. Biophye. Acta, 199, 447-452. Sommer, A. & Traut, R. R. (1974). Proc. Nat. Ad. Sci., U.S.A. 71, 39463950. Sommer, A. & Traut, R. R. (1975). J. Mol. Biol. 97, 471-481. Sommer, A. & Traut, R. R. (1976). J. Mol. Biol. 196, 995-1015. Staehelin, T., Maglott, D. & Monro, R. E. (1969). Cold Spring Harbor Symp. Quant. BioZ. 34, 39-48. St&filer, G. (1974). In Ribosonaea (Nomura, M., T&i&es, A. & Lengyel, P., eds), pp. 615 667, Cold Spring Harbor Laboratory Press, New York. Szer, W. & Leffler, S. (1974). Proc. Nat. Ad. Sci., U.S.A. 71, 3611-3615. Takanami, M. L%Okamoto, T. (1963). J. Mol. BioZ. 7, 323-333. Thomas, G., Sweeney, R., Chang, C. 8z Noller, H. F. (1975). J. il!foZ. BioZ. 95, 91-102. Sci., U.S.A. 72, Tischendorf, G. W., Zeichhardt, H. & St&ffler, G. (1975). Proc. Nat. Ad. 4820-4824. Van Duin, J. & Kurland, C. G. (1970). Mol. Gen. Genet. 109, 169-176. Wagner, R. & Gassen, H. G. (1975). B&hem. Biophye. Rea. Commun. 65, 519-529. Zamir, A., Miskin, R. & Elson, D. (1971). J. Mol. BioZ. 60, 347-364.
Chemical Inactivation of Escherichia coli 30 S Ribosomes with Maleic Anhydride : Identification of the Proteins Involved in Polyuridylic Acid Binding WCHAEL
CAXTRELL
AND
GARY R. CRAVEN
Laboratory of Molecular Biology and Department of Genetics University of Wisconsin Madison, Wise. 53706, U.S.A. (Received 7 February 1977) Modification of 30 S ribosomal subunits by the protein-modifying reagent maleic anhydride was found to inaotivate the particles for polyuridylic aoid binding. Reconstitution of 30 S ribosomes using 16 S RNA, maleylated total 30 S protein, and purified, unmodified proteins demonstrated that S4, Sll, S12, 813 and S18 are involved in poly(U) binding. Modified 30 S subunits contain all the ribosomal proteins and show normal sedimentation characteristics, indicating that the inaotivation is not simply due to the gross alteration of the particles. Correlation of these results with those of other workers is discussed.
1. Introduction In recent years a concerted effort has been directed toward understanding the functional roles of the ribosomal components. A number of approaches have been devised to determine the relative importance of the ribosomal proteins in specific functions. In light of the immense complexity of the Escherichia wli ribosome, it has become evident that definitive identification of the proteins involved in any function can only be made after similar results are obtained by a number of approaches. Thus several independent experimental approaches have been utilized to determine the components involved in messenger RNA and transfer RNA binding to the 30 S subunit. One of the early methods devised was the use of reconstitution experiments to study the effect on various activities of altering the component composition of the 30 S subunit (Nomura et al., 1969; Ozaki et al., 1969; Van Duin $ Kurland, 1970; Held et al., 1974). Another method employed protection studies to identify the components protected against chemical or enzymatic attack by the attachment of mRNA and tRNA (Noller et al., 1971; Rummel & Noller, 1973). A number of studies have been conducted to determine the ability of antibodies or antibody fragments specific for individual ribosomal proteins to block binding sites (Lelong et al., 1974; Sttiffler, 1974). Afllnity labels have been used to determine which proteins are physically located in the active sites (Pellegrini et al., 1974; Fiser et al., 1975; Pongs et al., 1975). More recently, chemical inactivation of the 30 S ribosome followed by reconstitution experiments has allowed identification of the components functionally involved in specific binding activities (Fanning, 1971; Thomas et al., 1975; Shimizu & Craven, 1976). 389
390
M. CANTRELL
AND
G. R. CRAVEN
We report here the application of chemical modification to identify the proteins at the site of inactivation for poly(U) binding. Modification of 30 S subunits is performed with maleic anhydride, a chemical reagent which primarily modifies free amino and sulfhydryl groups (Butler et al., 1967J969). This modification results in complete loss of poly(U) binding activity. We describe experiments in which reconstitution of 30 S subunits using 16 S RNA, modified 30 S total protein, and selected unmodified proteins allows us to identify proteins 54, Sll, 512, 813 and 518 as being at the site of inactivation for poly(U) binding.
2. Materials and Methods (a) Preparation
of ribosomes
and puti$ed
ribosornal
proteins
30 S ribosomes were isolated from E. coli MREGOO and purified as described previously (Craven 8.z Gupta, 1970). Ribosomal proteins were extracted from the purified 30 S subunits with 67% (v/v) acetic acid and fractionated by column chromatography with phosphocellulose (Mannix-P,, high capacity) following the procedure described by Hardy et al. (1969). In some cases the proteins were further fractionated by chromatography on carboxymethyl-cellulose (Whatman CM52) using the same buffers and elution conditions as applied in the phosphocellulose column chromatography. Identification of the proteins was established by a combination of chromatographic properties, 1 -dimensional polyacrylapolyacrylamide gel mide gel electrophoresis (Hochkeppel et al., 1976), and 2dimensional electrophoresis (Schendel & Craven, 1976). Sll and S9 were identified by immunodiffusion (Kahan et al., 1974) with antibodies supplied by L. Kahan and co-workers (University of Wisconsin). 30 S ribosomes labeled with 3H in wiwo were prepared by growing E. co&i MREBOO in lysine assay medium (Difco) containing [3H]lysine (New England Nuclear). Isolation of the ribosomes was as described above. (b) Male&
anhydride
modi$cation
of 30 S subunits
30 S subunits were heat-reactivated by incubation at 42°C for 20 min in the activation acetate, 0.2 Mbuffer of Zamir et al. (1971) : 50 mrvr-Tris.HCl (pH 7.2), 20 mM-mctgneSiUm NH,Cl, 2 mM-dithiothreitol. The ribosomes were then extensively dialyzed against poly(U) binding buffer (10 mivr-Tricine (pH adjusted to 7.6 with KOH), 20 m&r-magnesium acetate, KC1 to bring K+ concentration to 50 IIIM). The ribosomes remained in a heatreactivated state following this dialysis as defined by tRNA binding activity at 4°C. Maleic anhydride modification was then performed ss follows. 30 S subunits at 50 A,,, units/ml in poly(U) binding buffer were preincubated 2 min at 28”C, and the reaction was initiated by the addition of a solution of maleic anhydride (Aldrich) dissolved in dioxane (Aldrich, spectrophotometric grade) to give a molar ratio of maleic anhydride to 30 S subunits of 200 unless otherwise indicated. The volume of dioxane was always equal to or less than 5% of the volume of 30 S, an amount shown to have no effect on 30 S structure and adtivity (results not shown). Incubation was for 10 min at 28°C followed by addition of an excess of dithiothreitol. Stock [14C]maleic anhydride (Amersham-Searle) had a spec. act. of 21 mCi/mmol. (c) In vitro
functional
assay8
[3H]poly(U) binding assays were performed by the method of Smolarsky t Tal (1970) with the following modifications. Nitrocellulose filters (Millipore Corp. ; HAWP) were treated with 0.5 M-KOH for 30 min at room temperature. Unless otherwise indicated, 0.1 in 0.45 or O-9 ml of poly(U) binding buffer plus or 0.2 AzGO units of 30 S were incubated 1 maa-dithiothreitol with an excess of [3H]poly(U) (Schwarz-Mann; 11.5 to 22 mCi/mmol for 5 min at O’C). Approx. 0.5 pg of [3H]poly(U) per O-1 A,,, unit of 30 S was usually used. Samples were then filtered as described by Smolarsky & Tal (1970), and the dried filters were counted in 10 ml of a scintillation fluid composed of 5 g PPO and 0.1 g POPOP/l
305 PROTEINS
INVOLVED
IN POLY(U)
BINDING
391
toluene. Due to the variable polymer length of the [3H]poly(U), it is not possible to compute the absolute percentage of ribosomes active for poly(U) binding. The variations from Tables 1 to 4 in cts/min of [3H]poly(U) bound per Aaeo umt of 30 S ribosome are due to 2 factors. First, a number of different batches of [3H]poly(U) were used which had a 2-fold range in specific activities. Second, differences in chain length of poly(U) over the course of this study resulted in different numbers of cts/min bound per ribosome. The activity in the poly(U) assay was determined to be directly proportional to the ribosome concentration within each set of data. The poly(U)-directed [14C]phenylalanyl-tRNA binding activity of the ribosomes was measured by the method of Nirenberg & Leder (1964). About O-5 A,,, unit of 30 S particles w&9 assayed in 1 ml of a binding buffer containing 0.1 M-Tris*HCl (pH 7*2), 20 mM-magnesium acetate, 50 mm-KCl, 1 mnil-dithiothreitol, 40 pgpoly(U), 2.81 Azso units of total E. coli B tRNA (Schwartz-Mann) charged with [14C]phenylalanine (Amershsm-Searle, spec. act. 500 mCi/mmol Phe). The samples were incubated 10 min at 37”C, then diluted with 3 ml of the cold binding buffer and applied to a nitrocellulose filter which was washed twice with 3 ml of buffer. Filters were counted in 10 ml of a scintillation fluid composed of 5 g PPO, 60 ml Bio-Solv BBS-3 (Beckman) and toluene to 1 1. of total 30X protein and RNA (d) Exkzction Total 30 S protein and 16 S RNA were extracted from control and maleic anhydridemodified 30 S subunits by a modification of the procedure of Spicer & Craven (manuscript in preparation). The procedure allows extraction of the protein and RNA without exposing them to acidic conditions, which would partially reverse the mdeio anhydride modification (Butler et al., 1969; Glazer, 1976). A suspension of 305 subunits WMI dialyzed against the extraction buffer of 6 M-Urea, 60 mM-Tris*HCl (pH 7*6), 40 mM-magneSiUm acetate, 0.66 M-KCl, 2 mM-dithiothreitol at 4°C. Samples were incubated 1.5 h at room temperature with intermittent shaking and layered over 2 ml of 5% (w/v) sucrose in the extraction buffer in Beckman 50 Ti tubes. Samples were then centrifuged 15.5 h at 34,700 revs/min. The purified protein (supernatant) was dialyzed at 4’C verau8 5 M-Urea in RB2 buffer (30 mM-Tris*HCl (pH 7.6), 20 mM-UX3gUeSiUm acetate, 0.5 M-KCl, 1 mM-dithiothreitol). Portions were frozen and stored at - 70°C. The purified RNA pellet was redissolved in and dialyzed versus 10 mM-Tris*HCl (pH 7.6) and stored at -70°C. (e) Reconstitution and isolaGon of subunits With a few modifications, the reconstitution system described by Held et cd. (1973) was used. Proteins in 5 M-ureeRB2 buffer were dialyzed into RB2 buffer just prior to their use. The 16 S RNA was dialyzed into 30 mm-Tris+HCl (pH 7.6), 20 mna-magnesium acetate, 1 mM-dithiothreitol. RNA was then incubated 10 min at 42°C and the protein was added to the RNA at an appropriate volume to give the salt concentration of reconstitution buffer (30 ma/r-Tris*HCl (pH 7*6), 20 mM-magneSiUm acetate, 0.33 M-KC& 1 mxl-dithiothreitol). The incubation was continued for 60 min at 42°C. Unless otherwise indicated, reconstitutions were performed at a concentration of 1.5 A,,, units RNA/ml and at a molar ratio of protein to RNA of 2.5: 1. The molar ratio of individual proteins and the concentration of RNA were determined &9 described previously (Hochkeppel et al., 1976). Particles reconstituted from both modified and unmodified components showed normal sedimentation characteristics. After cooling to 4”C, the reconstituted particles were isolated by precipitation at 0°C with 0.5 vol. absolute ethanol (Staehelin et al., 1969; Thomas et al., 1975). The particles were resuspended in 0.4 to 0.6 ml of reconstitution buffer, and any undissolved materials were removed by low-speed centrifugation. Prior to testing for activity, the particles were then heated for 20 min at 42°C.
(f) Sucrose gradient centrifugation and polyacrylamide gel electrophoresis Samples containing 2 A,,, units of ribosomal subunits were layered on 3*5-ml 5% to 20% linear sucrose gradients in poly(U) binding buffer, centrifuged for 1.5 h at 55,000 revs/min at 4°C (Beckman SW56 rotor), and analyzed by pumping directly through a l-mm flow-through cell in a Beckman DB-G spectrophotometer. 26
392
M. CANTRELL
AND
G. R. CRAVEN
Two-dimensional gel electrophoresis was carried out essentially as described previously (Schendel & Craven, 1976). A total of 15 to 30 A 280 units of 30 S ribosomes was dialyzed into 6 M-urea, 0.03 M-Tris (pH 7*6), 5 mM-dithiothreitol and treated with pancreatic ribonuclease prior to electrophoresis. The proteins were separated in the first dimension by disc gel electrophoresis as described by Howard & Traut (1973) for 1 h at 3 mA/tube, then 5 to 7 h at 6 mA/tube. The proteins were separated in the second dimension by slab gel electrophoresis as described by Martini & Gould (1971) with slight modifications. The 2-dimensional gel apparatus was identical to that of Mets & Bogorad (1974). The first dimension gels were dialyzed at 37°C against: (1) 1 M-phosphate (pH 7-O), 1.0% (w/v) sodium dodecyl sulfate (30 min) ; (2) 0.01 M-phosphate (pH 7*1), l.Oo/o sodium dodecyl sulfate (20 min) ; and (3) 0.01 M-phosphate (pH 7*1), 0.10% sodium dodecyl sulfate (25 min) in preparation for the second dimension. The second dimension gel solution was identical to that of Martini & Gould (197 1) except the amount of N,N-methylene bisacrylamide was doubled. After polymerizing the l-dimensional gels onto the second dimension slab, electrophoresis proceeded for 15 to 16 h at 60 V.
3. Results (a) Modijimtion
of 30 X subunits with muleic anhydride
Figure 1 shows that the reaction of 30 S subunits with maleic anhydride results in the loss of both poly(U) and Phe-tRNA binding activities. Concomitantly, there is modification of the 30 S subunit as measured by the uptake of [14C]maleic anhydride (Fig. 2). There are three possible explanations for this inactivation : (1) loss of activity is due to direct inactivation of the site for poly(U) binding; (2) modification of the subunit results in a loss of ribosomal proteins, thereby causing inactivation; (3) modification causes a conformational change in the subunit which leads to loss of activity.
Moles mleic anhydride added/3OS subunit
FIG. 1. The effect of meleic anhydride on poly(U) and Phe-tRNA binding activity. The 30 S subunits were modified as described in Materials and Methods with varying amounts of maleio anhydride. Numbers on the abscissa represent the ratio of maleic anhydride to subunit in the reaction mixture. The reaction was terminated with an excess of dithiothreitol and assayed and poly(U)-directed phenylalanyl-tRNA binding directly for poly(U) binding (-•--~--) (-o-o-). A total of 0.47 &so unit of 30 S was used per assay sample for the sH-labeled poly(U) binding assay, and 100% activity corresponded to 8320 cts/min of 3H radioactivity. A total of 0.49 A,,, unit of 30 S was used per assay sample for the 14C-lebeled poly(U)-directed phenylalanyl-tRNA binding assay, and 100% activity corresponded to 2232 ots/min of 14C radioactivity.
305
PROTEINS
INVOLVED
IN
POLY(U)
BINDING
393
Moles maleic anhydride addedl3OS submit
Fm. 2. Uptake of [14C]maleio anhydride into 30s subunits. Subunits were modified as described in Materials and Methods with varying amounts of [l*C]maleic anhydride at a spec. act. of 1.388 mCi/ mmol. The reaction was terminated with an excess of dithiothreitol, and portions of 0.93 A 280unit were diluted to 2 ml with the reaction buffer. The samples were subsequently filtered on Millipore filters, washed, and counted as described in Materials and Methods. The plateau level of 8100 cts/min bound corresponds to 40 mol of maleic anhydride bound to each mol of 30 5. Backgrounds for any 14C cts/min of maleic anhydride products bound to Millipore filters in the absence of 30 S were subtracted.
Although identification of the 30 S components involved in the inactivation would yield valuable information no matter what the mechanism of inactivation, we were interested in determining which of the three potential mechanisms might be involved in the inactivation. Therefore, we initiated a series of studies to determine if the protein composition and/or the conformation of the modified particles had been altered. (b) Determination of the protein composition of modi$ed subunits To determine if any of the 30 S proteins substantially dissociate from modified subunits, 30 S particles were modified by maleic anhydride and isolated by centrifugation through a layer of 5% (w/v) sucrose in poly(U) binding buffer. Isolated control particles and isolated maleylated particles were analyzed for protein content by twodimensional gel electrophoresis as shown in Figure 3. Although the mobilities of many of the proteins are altered after maleylation due to the addition of negatively charged maleic acid groups, there does not appear to be any substantial loss of any protein as a consequence of the chemical modification. No conclusion can be drawn concerning the presence of 821 as it usually fails to appear in the gel system we have employed. Quantitation of the two-dimensional gels was not possible due to the alterations in electrophoretic mobility. As a result, it was possible that there could have been some general loss of ribosomal protein. To determine if this was the case, in &o-labeled ([3H]lysine) 30 S ribosomes were modified with maleic anhydride and centrifuged through sucrose as described above. Supernatant fractions and the pellet were then analyzed to determine the amount of [3H]protein in each. For control 30 S subunits and modified 30 S subunits, respectively, it was found that only 1.4% and 1.7% of the [3H]proteins were present in the supernatant fractions. It therefore appears that there is no substantial change in the protein composition of modified subunits.
394
M. CANTRELL
AND
G. R.
CRAVEN
FIG. 3. Two-dimensional gel electrophoresis of 30 S ribosomal proteins from isolated control (a) and isolated modified (b) 30 S subunits. (aI) Acidic protein, (aa) basic protein from control 30 S subunits. (b,) Acidic protein, (b,) basic protein from modified 30 S subunits.
(c) Mod$cation
does not cawe any large conformational change
It was also possible that maleic anhydride modification induced a conformational change in the particle, which in turn produced a loss in poly(U) binding activity. To test this possibility, modified and unmodified 30 S subunits were analyzed on sucrose gradients and the sedimentation constants determined. Figure 4 shows that the particles have normal sedimentation constants both before and after modification, even up to a ratio of 450 mol of maleic anhydride added per mol of 30 S present. The data therefore indicate that there are at least no gross alterations in the 30 S conformation. We therefore tentatively suggest that chemical derivatization of the 30 S subunit under these conditions leads to a loss of 90% of the poly(U) binding activity by direct
30s
PROTEINS
INVOLVED
IN
l?OLY(U)
BINDING
395
I Top -
Bottom Fractions
Fra. 4. Sedimentation of control 30 S subunits (a) and modified 30 S subunits (b). Modification was carried out as described in Materials and Methods at a ratio of 450 mol of maleic anhydride added per mol of 30 S. Samples containing 2 A,,,, units of 30 S in a volume of 46 ~1 were layered on sucrose gradients made up in the poly(U) binding buffer and centrifuged aa described in Materials and Methods.
modification of the components involved in that binding. This suggestion is further supported by the finding that modification of poly(U)-30 S complexes with maleic anhydride results in no loss of bound poly(U). (d) The protein component is modi$ed by maleic anhydride To determine whether maleic anhydride modifies the RNA or protein moieties of the 30 S subunit, a suspension of 30 S subunits was treated as described in Materials and Methods with a ratio of 228 mol of [14C]ma 1eic anhydride per mol of ribosomes in the reaction mixture. The protein and RNA were subsequently separated (see Materials and Methods), and each was dialyzed extensively versus its post-centrifugation extraction buffer and analyzed for [14C]maleic anhydride incorporation. Under these conditions, greater than 99% of the bound maleic anhydride remained associated with the protein fraction while less than 1% remained associated with the RNA fraction. We conclude that most, ifnot all, of the modification takes place on the protein component of the 30 S subunit. In support of this, two-dimensional gel electrophoresis of proteins extracted from maleic anhydride-inactivated ribosomes indicates that many of the proteins have altered electrophoretic mobilities due to modification (compare Fig. 3(a) and (b)). (e) Determination of the 30 S protein as the site of inactivation Heterologous reconstitution experiments were performed to determine whether the site of maleic anhydride inactivation for poly(U) binding was in the protein or the
396
M. CANTRELL
AND
G. R.
CRAVEN
RNA moiety of the 30 S subunit. The 16 S RNA and 30 S total protein were isolated from control and modified subunits. Reconstitutions and isolations were then performed with the combinations shown in Table 1, followed by measurement of the poly(U) binding capacity of each reconstituted particle. The site of inactivation is seen to be completely localized in the protein moiety. TABLE
Ident@c&on
of the protein moiety as the site of inactivation
source of RNA
c M c M
1
30s 305 30s 305
source of protein
c c M M
30s 30s 305 30s
Control activity after reconstitution (%I 100 98 19 17
RNA and total protein were extracted from unmodified (C 305) and modifled 305 subunits (M 305). Reconstitutions were then performed on all pairwise combinations with 2.6 molar equivalents of total protein per molar equivalent of RNA added, and the particles were isolated and assayed for poly(U) binding as described in Materials and Methods. The 100% activity represents the activity of particles reconstituted from unmodified protein and RNA. Its average value wss 8360 cts/min per 0.1 Ass,, unit of 305.
(f) Identi$cution
of the individual
proteins inuctivated by maleic anhydride
In order to identify the proteins at the site of inactivation, a series of reconstitution experiments were undertaken in which 16 S RNA and modified total protein were reconstituted in the presence of selected unmodified proteins. Preliminary experiments showed that the addition of more than one unmodified protein to the reconstitution mixtures of 16 S RNA and modified total protein was required to give an appreciable return of poly(U) binding activity. As a result, the 30 S proteins were divided into three groups to be studied for their ability to restore binding activity. Group 1 was composed of proteins Sl, S2, 55, 58, S9, Sll, S20 and S2I ; the composition of group 2 was proteins 53, 54, 512, 513, 514, 515, 518 and S19; and group 3 consisted of proteins S6, S7, SlO, S16 and 517. Table 2 presents averages of a number of experiments and shows that inclusion of either group 1 or group 2 proteins in the reconstitution mixture produced 30 S ribosomes with substantial poly(U) binding activity while the ribosomes obtained upon addition of group 3 proteins showed no poly(U) binding activity. Thus proteins S6, 57, SIO, S16 and 517 were eliminated as possible components of the site of maleic anhydride inactivation. It should be noted that the values for poly(U) binding given here are relative values for the activity of the particles and do not reflect the absolute percentage activity of the ribosome. The high percentage activity returned may either be a consequence of the variability commonly seen in reconstitution systems (Held et al., 1973) or a reflection of the fact that some of the ribosomal proteins have fractional stoichiometries (Craven et al., 1969 ; Hardy, 1975). All assays were determined to be strictly linear over the ranges used (see Materials and Methods). To determine which proteins within groups 1 and 2 were important for restoration
30s
PROTEINS
INVOLVED
IN
POLY(U)
BINDING
397
of poly(U) binding activity, a series of experiments was performed on each group separately in which the effect of the deletion of selected proteins from a group was ascertained (Table 3). Examination of Table 3 shows that the values obtained for percentage activity returned by different sets of proteins can be grouped into two distinct categories; those with activities close to lOO%, and those with activities much lower than 100%. TABLE
2
Restoration of poly( U) binahg activity by selectedprotein groups Proteins present during reconstitution MTP MTP MTP MTP MTP
+ + + +
control total protein group 1 proteins group 2 proteins group 3 proteins
Activity
returned (%) 0 100 83 172 0
Reconstitutions were performed on mixtures containing 1 molar equivalent of 16 S RNA plus 2.6 molar equivalents of modified tote1 protein and 2.6 molar equivalents of the indicated unmodified proteins. The reconstituted particles were then isolated and essayed for poly(U) binding activity. Group 1 contains proteins Sl, 52, S5, S8, SQ, Sll, S20 and S21; group 2 cont&s proteins 53, 54, 512, 513, 514, S16, 518 and SlQ; and group 3 contains proteins S6, 57, SlO, S10 cmd S17. The average activity for the 100% value was 3600 cts/min per 0.2 Aaso unit, end the average activity for the 0% value wss 310 cts/min per 0.2 Azao unit.
More specifically, there is a set of unmodified proteins whose deletion from the reconstitution results in activities within 22% of control, and there is a set whose deletion results in a reduction of activity by more than 54%. We suggest that the proteins directly required for poly(U) binding activity fall in the latter category. Table 3A thus shows that proteins Sl, 52, S5,58,520 and S21 can be eliminated as possible components at the site of maleio anhydride inactivation. Similarly, Table 3B shows that proteins S3,514,515 and S19 can be eliminated from group 2. As a result of the above studies, the proteins potentially involved in poly(U) binding were narrowed down to 512 and/or 513, 54 and/or 518, and Sll and/or S9. However, on the basis of those studies it could not be determined whether one or both of the proteins in each of these three pairs is important for activity. Final identification was thus done by reconstitutions using each protein in a highly purified form. Table 4 shows that deletion of any one of the implicated proteins except S9 results in a reduction in poly(U) binding activity. We therefore conclude that proteins 54, Sll, S12,513 and 518 are the major components of the site of inactivation for poly(U) binding. In addition, these proteins may directly be involved in the poly(U) binding site. We cannot, however, rule out the possibility that their modification may involve a small, undetectable conformational change in the subunit. 4. Discussion In a previous paper (Shimizu $ Craven, 1976) we reported the chemical inactivation of the 30 S ribosome for Phe-tRNA and fMet-tRNA binding by iodination. In that
398
M. CANTRELL
AND
G. R.
TABLE
CRAVEN
3
Restoration of activity by group 1 and group 2 proteins Proteins
present during reconstitution
Relative
A MTP MTP MTP MTP MTP MTP MTP MTP MTP
+ + + + + + + +
Sl, -, Sl, Sl, Sl, Sl, Sl, Sl,
52, SS, S8, S9, S2, SS, 58, S9, -, S5, S8, S9, 52, -, 58, S9, S2, S5, -, S9, 52, 55, S8, -, S2,55,58, S9, 52, S5, 58, S9,
Sll, Sll, Sll, Sll, Sll, -, Sll, Sll,
Expt 1 0 100
S20, S21 520, S21 S20,521 S20,521 520, S21 520,521 -, S21 520, -
96 127 40
B MTP MTP MTP MTP MTP MTP
+ + + + +
53, S4,512,513, 53,54,512,513, -, 54, S12,513, S3,--,S12,513, S3,S4, -, -,
S14, S15,518, -, -, S18, -, -, S18, -, -, -, -, -,S18,
S19 -
Expt 1 0 100 7.5
activity
returned
94 75 46 79 85
96 84
Average 0 100 94 113 95 101 43 88 85
Expt 2 0 100 95 78 36 36
Expt 3 0 100 93 80 48 54
Average 0 100 88 79 42 45
Expt 2 0 100 111
Expt 3 0 100
(%) Expt 4 0 100 76 113
Reconstitutions, isolations, and assays were performed as described in the legend to Table 2 and Materials and Methods. The average activity in A for the 100% value was 4630 cts/min per 0.2 in B for the A 260 unit and 310 cts/min per 0.2 A,,, unit for the 0% value. The average activity 100% value was 2930 cts/min per 0.2 A,,, unit and 460 cts/min per 0.2 A,,, unit for the 0% value. TABLE
Identi$cation of the proteins required Proteins present during reconstitution
4
for poly( U) binding activity Activity
returned 1%)
MTP MTP + control total protein MTP + S4, S9, Sll, 512,513,518 MTP + -, S9, Sll, 512, S13,518
0 100 118 82
(100) (69)
MTP MTP MTP MTP MTP
111 79 56 76 81
(94) (67) (47) (64) (69)
+ + + + +
54, -, Sll, 512, S13, S18 S4, S9, -, 512,513, S18 54, S9, Sll, -, 513, S18 S4, S9, Sll, 512, -, 518 Sr, S9, Sll, S12, 513, -
Reconstitutions, isolations, and assays were performed as described in the legend to Table 2 and Materials and Methods. The average activity for the sample containing 16 S RNA plus modified total protein and control total protein was 12,400 cts/min per 0.1 Azso unit. The average activity for the sample containing 16 S RNA plus modified total protein w&s 1700 ots/min per 0.1 A,,, unit. The numbers in parentheses are the rtotivity restored normalized to the activity of MTP plus the total complement of tested proteins.
30s
PROTEINS
INVOLVED
IN
POLY(U)
BINDING
399
study, reconstitution experiments analogous to those described here identified proteins 53,514 and 819 as being involved in fMet-tRNA binding and proteins Sl, S2, S3, S14 and S19 as being involved in Phe-tRNA binding. In this study we have extended that type of approach to identify the 30 S ribosomel proteins involved in poly(U) binding. Chemical modification with the protein-modifying reagent maleic enhydride has been shown to completely inactivate the 30 S ribosome for both poly(U) and PhetRNA binding. We have found by reconstitution that proteins 84, Sll, 512, 513 and 518 are critical proteins in returning this poly(U) binding activity. As Table 4 shows, it is probable that 512 is the most essential protein of the five proteins implicated. The observation that modification of amino groups by mctleic anhydride results in loss of poly(U) binding is not, by itself, strong evidence that amino groups are directly involved in poly(U) binding. However, Chang BECraven (manuscript in preparation) have surveyed a wide range of protein-modifying reagents and found that the only reagents inactivating the 30 S subunit for bacteriophage Qfi natural mRNA binding are either reagents which can cross-link the ribosome or which modify amino groups (with the exception of Rose Bengal, a histidine-derivatizing reagent). The amino acids modified in that study, which utilized over 25 protein-derivatizing reagents, include arginine, tyrosine, tryptophen, cysteine, lysine and the carboxyl groups of glutamate and asp&ate. There is thus a strong possibility that lysine amino groups are selectively involved in mRNA binding. It has been suggested that poly(U) binding to the ribosome is not specific (Nomura, 1970; Nomurrt & Held, 1974). Such suggestions are based partially on the fact that basic proteins alone can bind poly(U). However, there are a number of lines of evidence suggesting that the binding of poly(U) to the ribosome is specific. The earliest evidence for the specificity of poly(U) binding was the determination that poly(U) binds only to the 30 S subunit and not to the 50 S subunit (Takanami & Okamoto, 1963). It has been known for some time that aurintricarboxylic acid inhibits both natural mRNA and poly(U) binding to the ribosome (Grollman & Stewart, 1968). In addition Szer & Leffler (1974) have shown that poly(U) competes for MS2 RNA binding. We have found that prior binding of Q/3 RNA to unwashed 30 S ribosomes competes for the poly(U) binding capacity of those ribosomes (Cantrell & Craven, unpublished results). We would thus suggest that poly(U) and natural mRNA compete for the same site on the 30 S ribosome. Wagner & Gassen (1975) have shown that the oligonucleotide U-U-U-(nhr)2’U can direct Phe-tRNA binding while covalently bound to the 30 S ribosome. However, the most compelling indication of specificity of poly(U) binding comes from the identification of the proteins involved. If the binding were non-specific one might expect poor correlation with the results obtained using other mRNAs. However, this is not the case. The proteins found by our experiments to be involved in the poly(U) binding site are a distinct group of five proteins, which have been implicated in mRNA binding by a number of other approaches, using different mRNAs. One of these approaches has involved the use of chemically reactive mRNA analogs or affinity labels. Pongs e.tal. (1975) used this approach to identify the 30 S ribosomal proteins modified by attachment of a bromolated derivative of the AUG codon to 70 S and 30 S ribosomes. The proteins they have identified coincided exactly with our proteins, 54, Sll, 512,513 and X18, with the addition of S21. Similarly, Ltihrman et al. (1976) utilized an mRNA affinity label, G-U-U-iacn5U, and found attachment of Sl, S18 and 512. Fiser et al. (1975) implicated proteins Sl, 518 and 521 by the photoaffinity reaction of poly(4-thiouridylic acid) with 70 S ribosomes.
400
M. CANTRELL
AND
G. R. CRAVEN
Chemical modification studies have also produced much valuable information. Derivatization of 30 S ribosomes with Rose Bengal followed by reconstitution has implicated S2 and 53 to be involved in poly(U) and Phe-tRNA binding (Thomas et aE., 1975). However, there was only a partial inactivation (approx. 65%) of the poly(U) binding in this study. Chang & Craven (manuscript in preparation) have used the reagent 2-methoxy-5nitrotropone to identify Sl, 512, 513 and S21 as proteins essential for phage Q/I RNA binding. Reconstitution experiments by Nomum’s group have shown 811 and 512 to be very important for the ambiguity and fidelity of mRNA translation (Ozeki et al., 1969; Nomura et al., 1969). More recently they have also shown protein 512 as well as 16 8 RNA to be involved in determining the speci6city of initiation (Held et al., 1974). We have not found a requirement for unmodified Sl in our system. However, we would like to emphasize that this does not constitute a discrepancy with the many studies pointing to the importance of Sl in mRNA binding (Van Duin & Kurland, 1970; Noller et al., 1971; Piser et al., 1975). It may be that the necessary functions of Sl are merely not inactivated by maleic anhydride under our conditions. Structural studies of the 30 S ribosome suggest that the identified proteins may indeed be situated physically close to each other. Sommer & Traut (1974,1975) have produced cross-links of 54 and 513 and of 54 and S12. Recent work by that group (Sommer & Traut, 1976) has also identified 512 and 513 plus Sll and 513 as crosslinked products obtainable from the 30 S ribosome. In addition, immunoelectron microscopy (Lake & Kahan, 1975; Tischendorf et al., 1975) has shown that antigenic sites for 54, Sll, 512, 513 and 518 are in close proximity on the head region of the ribosome. The model produced by Stijffler and co-workers is shown in Figure 5 with selected antigenic sites for the proteins commonly implicated as being within the mRNA binding site. Due to the highly complex nature of the ribosome, independent information from a wide variety of experimental approaches is needed to unequivocally identify the
(a)
(b)
FIQ. 6. Model of the 30 S ribosomal subunit based on immunoelectron microscopy (Tischendorf et al., 1976). Two views are shown, giving selected antigenic sites for the ribosomel proteins commonly implicated in mRNA binding. The proteins identified in this study are circled by solid lines.
305
PROTEINS
INVOLVED
IN
POLY(U)
BINDING
401
components involved in mRNA binding. It is therefore encouraging to note the overwhelming agreement found by several independent techniques for a small group of ribosomal proteins. This combination of structural and functional information should ultimately give a much more coherent picture of the ribosome. The authors would like to offer special thanks to Dr T. Fanning whose preliminary studies provided the basis for this work. We are also indebted to Dr L. Kahan and D. Winkelmann for their help in immunochemical experiments and to MS C. Bloomer for providing purified ribosomal proteins. This work was supported by the Graduate School and the College of Agricultural and Life Sciences, University of Wisconsin, Madison and by research grant GM15422 from the National Institutes of Health. One of us (M. C.) was supported by training grant GM01874 from the National Institutes of Health. We dso acknowledge use of the University of Wisconsin Biochemistry Department pilot plant, directed by Dr J. Garver and supported by the United States Public Health Service grant Fr-00214.
Butler, Butler,
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