The Transfer RNA Binding Site of the 30 S Ribosome and the Site of Tetracycline Inhibition

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1 The Transfer RNA Binding Site of the 30 S Ribosome and the Site of Tetracycline Inhibition GARY R. CRAVEN, RAY GAVIN, AND THOMAS FANI~IING Laboratory of Molecular Biology and Department of Genetics, University of Wisconsin, Madison, Wisconsin Investigations from a number of laboratories (Hardy et al., 1969; Moore et al., 1968; Fogel and Sypherd, 1968; Kaltschmidt et al., 1967) have demonstrated that Escherichia coli ribosomes are composed of a large number (probably 50 to 55) of chemically distinct proteins. The experiments reported earlier in this volume by Nomura et al. have shown that for the 30 S ribosomal subunit each individual protein makes a measurable contribution to the final ability of the particle to function. Thus the ribosome is a highly complex organelle whose function is intricately dependent on the overall set of interactions of its many components. What is not clear from these results is the question of which proteins should be designated as being directly and intimately responsible for specific functions. It is of basic importance to determine which of the 20 proteins in the 30 S particle are involved in the actual binding of aminoaeyl transfer RNA (trna) and of messenger. The data of Nomura et al. (this volume) indicate that a number of proteins when deleted yield particles incapable of carrying out the trna binding activity. It is difficult to decide which of these proteins are personally responsible for the formation of this binding site. There have been two fundamentally different approaches to this problem taken in this laboratory. The first has been to probe the site of action of the antibiotic tetracycline. Experiments by several workers have suggested that tetracycline's site of inhibition is at the trna binding locus of the 30 S particle (Suzuki et al., 1966; Sarkar and Thach, 1968). Should this be true, a reasonable approach to the identification of the ribosomal protein(s) involved in trna binding is to select for mutant strains resistant to the action of tetracycline and to ultimately identify the protein(s) altered as a result of the mutation. The experiments designed along these lines have yielded a strain of E. coli resistant to very high levels of tetracycline (250 /~g/ml). This strain has been shown to contain a protein synthesizing system resistant to tetracycline inhibition in vitro. This resistance, which resides in the ribosome, can be co-transduced with the AroE locus at a high frequency and therefore maps in the general region associated with ribosomal proteins. 129 MESSENGER AND trna BINDING SITE l GROUP SELECTIVE REAGENT \ 9 INACTIVE BINDING SITE DISSOCIATION AND RECONSTITUTION WITH ACTIVE PROTEINS FIOURE l. A diagrammatic representation of ribosome inactivation using group specific derivatizing reagents. The second approach taken to the general problem of ribosomal binding sites has been to utilize selective group protein derivatizing reagents with the intent of specifically inactivating the 30 S subunit for trna binding. Similar experiments were initiated by Traut and Haenni (1967). The basic rationale of this method is schematically drawn in Fig. 1. In this illustration the ribosome is pictured as a collection of different proteins only several of which are actually responsible for recognition of messenger and trna. A reagent is pictured as reacting with a specific functional group on all proteins available to the environment, one or more of which are the trna binding site proteins. Modification of the binding site proteins is assumed to inhibit directly the binding function, and if this is in fact the case then identification of the proteins involved can be achieved by dissociation and reconstitution with active proteins using the techniques described by Traub and Nomura (1969). The drawing in Fig. 1 points out one difficulty in this general approach. It is conceivable that one of the proteins distal to the actual binding locus and not involved directly in binding could be identified erroneously as one of the binding proteins.

2 130 CRAVEN, GAVIN, AND FANNING (I) R-SH+ C(NO=)4 ) R-SNOt-I- C(NO=)~+ H*, RLSH R-S-S-R'+ H NO~ activation reaction has been achieved by the presence of either poly U, trna or both together. RESULTS OH O" O (2) + C(NOz)4 + C(N02)3+ 2H 'P R R FmuRE 2. Proposed mechanisms for the reactions of tetranitromethane with the sulfhydryl and phenolic residues of proteins (modified from Sokolovsky et al., 1966). This could happen if modification of this protein by the reagent produced a product which prevented proper reconstitution yielding an inactive particle. To avoid this possibility it is essential that any protein identified as residing in the binding site must be protected from inactivation when reaction with the reagent is conducted in the presence of substrate, in this case trna and messenger. An initial screening of potentially useful reagents resulted in the finding that tetranitromethane (TNM) was extremely effective in inactivating purified 30 S particles for phenylalanine 'trna binding directed by poly U. Tetranitromethane, as described by Sokolovsky, Riordan, and Vallee (1966) modifies tyrosines in proteins by the reaction shown in Fig. 2. In addition to the nitration of tyrosine, this reagent can also oxidize cysteine presumably to give a disulfide bridge as shown in Fig. 2. A third possible side reaction this reagent may promote is not shown in Fig. 2, but involves the presumed intermediate in the nitration of tyrosine, namely a quinone structure (Doyle, Bello, and Roholt, 1968). If the quinone is the immediate oxidation product it is open to ortho-directed substitution with any closely available nucleophilic group. When tetranitromethane is used in high molar excess, then nitration at the ortho-position is the principal product. If low molar excesses are used, then presumably linkage could be achieved with nearby functional groups such as amino groups and sulfhydryl groups. This type of reaction might then lead to covalent cross-linkage of proteins located next to one another on the ribosome. Such reactions are being investigated in order to learn more about the three dimensional relationships of the various ribosomal proteins. In any event, tetranitromethane has been found to inactivate the 30 S particle under the condition of high molar excess. In addition, protection from this in- ~NACTIVATION BY TETRANITROMETHANE Kinetics of inactivation and nitrotyrosine formation. The conditions for tyrosine nitration and enzyme inactivation by tetranitromethane have been well described (Sokolovsky et al., 1966). In our investigations of 30 S ribosome dependent binding of phenylalanine trna and poly U, essentially identical conditions for inactivation have been employed as those which are optimal for other systems. We have found that at a ph of 8.0, a molar excess of 600 (moles of tetranitromethane per mole of 30 S ribosome), and a temperature of 28~ rapid and reproducibly complete inactivation occurs within 30 rain. This is shown in Fig. 3. Also presented in this figure is a time dependent formation of nitrotyrosine as determined by amino acid analysis. The number of tyrosines nitrated after 60 rain of incubation approaches 25 (out of a potential 50 tyrosines) per 250,000 daltons of ribosomal protein. Thus the degree of inactivation has roughly the same kinetics as does the appearance of nitrotyrosine. Experiments not shown in Fig. 3 indicate that in addition, the formation of nitroformate, as measured by the absorbanee at 420 mf, has kinetics close to that of inactivation. However, I00 ao o~ > a ~- o o ~ 20 I0 (o o e- l- I TIME (MIN.) FIO,m~ 3. The time course of inactivation of 30 S ribosomes by tetranitromethane. The ribosomes were incubated at 28~ with a 662-fold excess of TNM. At various times atiquots were taken and assayed for either 14C- Phe-tRNA binding by the method of Kurland (1966), or for tyrosine --II-- I- by a modification of the procedures of Spackman et al. (1958). w w

3 BINDING SITE OF THE 30 S RIBOSOME [31 the amount of nitroformate produced by the reaction appears to be somewhat in excess of the number of tyrosines nitrated. That this discrepancy can be accounted for by SH oxidation has been suggested by titration studies using Ellman's reagent (Ellman, 1959). Other studies to be reported elsewhere have suggested that it is possible to preferentially carry out the oxidation of the SH groups by performing the reaction at ph 6.0. Ribosomes modified under these conditions maintain at least 80% activity, allowing the conclusion that the principal mechanism of inactivation hy TNM must involve nitration or possibly crosslinking of tyrosine groups. Protection from TNM inactivation. As mentioned in the introduction, an important criterion to be applied to any reagent capable of inactivation is that the process be inhibited by the presence of substrate. To meet this requirement, 30 S ribosomes were incubated with TNM under optimal conditions at two different concentrations of Mg ++. One concentration, 20 mm, was that routinely used, as it is optimal for poly U directed Phe-tRNA binding. The other, 0.5 mm Mg ++, was sufficiently low that neither poly U nor Phe-tRNA bind significantly. Ribosomes maintained at either of these two Mg ++ concentrations undergo identical rates of inactivation by TNM, as is seen by comparing Fig. 4 with Fig. 3. However, when either poly U, trna, or both are included in the reaction mixtures, significant protection from the inactivation by TNM is observed only at 20 mi Mg ++. Fig. 4 (d), on the other hand, shows that 16 S ribosomal RNA has no protective effect. It is important to point out that the assay used to measure binding activity is necessarily basically different in the protection experiment of Fig. 4 (b and c). In this experiment, nonradioactive trna is bound first to the ribosome during the incubation with TNM; at various times additional trna is added which is charged with radioactive phenylalanine. The Phe-tRNA then exchanges with previously bound trna to become bound. Using this type of exchange assay, only about 25% as much Phe-tRNA can be bound. Therefore one must take this experiment with the reservation that one cannot be certain that this exchange assay truly measures the same binding site that the more direct binding assay measures. A more definitive protection experiment is presently being conducted which involves act,!al stripping of bound trna and poly U after the TNM reaction and then directly measuring the binding activity. The protection experiments outlined above allow one to rule out a possible objection to the TNM inactivation phenomenon itself. Although the reactions of TNM have been extensively characterized with references toproteins, little is known about I00, I001~ (c) ~ I (d) rrna ~60 7- _ 6ol F- '' # 40 # m M Ma =" 20 mm Itlg" J I i i I I TIME (MIN) TIME (MIN.) i l I I l I Fmu~ 4. Inactivation of 30 S ribosomes preincubated with Poly U, trna, or rrna. The 30 S ribosomes (8.10D~60 units) were incubated with (a) 100 #g Poly U, (b) 25 OD2~ 0 units deacylated trna, (c) 100/~g Poly U plus 25 OD2c 0 units deacylated trna or, (d) 2.5 OD260 units 16 S rrna in buffer containing either 20 mm Mg ++ --Q--C)-- or 0.5 mm Mg After a 10 rain preincubation, tetranitromethane was added to give a 593-fold excess over 30 S ribosomes. Incubation was continued, and aliquots of the reaction mixture were removed at the indicated times and assayed for 14C-Phe-tRNA binding activity.

4 132 CRAVEN, GAVIN, AND FANNING the potential reactions with nucleic acids. Thus it is conceivable that in the normal inactivation experiments, the TNM is not directly inhibiting ribosome function, but residual reagent is attacking the Phe-tRNA or poly U. This was always guarded against by the addition of excess mercaptoethanol at the termination of the reaction which should reduce all residual TNM. In addition, the fact that the inactivation is time dependent argues against this explanation. However, since the protection experiments involve incubation of trna and of poly U in the presence of TNM, they can be used as evidence against the possibility that TNM directly attacks the substrate. Finally, experiments to be reported elsewhere have shown that trna, poly U and 16 S ribosomal RNA do not cause the production of significant amounts of nitroformate. Furthermore, preincubation of poly U and PhetRNA with TNM does not cause any loss of their subsequent abihty to bind to the ribosome. Reconstitution o f inactivated ribosomes. Although 16 S ribosomal RNA has no pronounced ability to reduce TNM with the production of nitroformate, it is conceivable that tb.e loss of ribosome function is due to a specific reaction with the RNA component rather than the protein. To rule this possibility completely out, reconstitution experiments were conducted using the technique of Traub and Nomura (1969). Figure 5 presents a reconstitution experiment in which 16 S RNA from non-nitrated ribosomes is mixed with an equivalent amount of protein extracted from this same ribosome preparation. In addition, varying amounts of protein extract are included in the reconstitution mixture which was derived from ribosomes previously inactivated with TNM. As can be seen, the amount of activity of the ribosomes reconstituted in this fashion is directly dependent on the amount of protein extracted from nitrated ribosomes. In fact, the per cent activity of the reconstituted particles is almost precisely that expected if a single component of the inactive protein mixture is responsible for the loss of activity. That is, the particles have almost 50% activity when active and inactive proteins are present in equivalent amounts. Similarly, when one-third of the protein mixture was derived from active ribosomes, and two-thirds from TNM-treated ribosomes, the particles were only 33% as active as the control. However, final evidence that TNM's site of inactivation is a single compopent must await ultimate purification. More important for immediate considerations is the suggestion that TNM's site of inactivation is the ribosomal protein rather than the ribosomal RNA. This conclusion is more directly implied by the result that 16 S ribosomal RNA extracted from nitrated ribosomes is eom- I- I- I B lo I I I I 9 5 I EQUIVALENTS OF NITRATED PROTEIN FmURE 5. An experiment demonstrating that the 30 S ribosomal component(s) inactivated by tetranitromethane resides in the protein fraction. Four identical reeonstitution mixtures containing 16 S rrna and 30 S protein from untreated 30 S ribosomes were prepared by tile method of Traub and Nomura (1969). To these reconstitution mixtures were added the indicated amounts of protein prepared from 30 S ribosomes that had been inactivated by tetranitromethane. Following reconstitution, the ribosomes were collected by eentrifugation and assayed for their ability to bind I~C-Phe-tRNA. pletely active when reconstituted with untreated ribosomal protein. Traub et al. (1967) have reported the isolation and functional characterization of the split proteins produced by CsCl treatment. Two of these proteins appear to be essential for Phe-tRNA-poiy U binding when excluded from the reconstituted particles. Thus it is reasonable to inquire if the site of TNM inactivation is identical with either or both of these split proteins. To this end, reconstitution experiments were conducted using inactive total protein and active split proteins or core proteins. From the curves in Fig. 6 it is apparent that the principal locationofthe active protein component(s) resides in the core fraction--although some small

5 BINDING SITE OF THE 30 S RIBOSOME > 20 t) 10 two bands in the lower portion of the pattern. However, gel patterns of the pellet supernatant do not reveal these two bands (although several other bands higher up in the gel profile are present in relatively small amounts). Thus TNM treated ribosomes do not appear to be losing significant amounts of any proteins. It is clear, however, that at least two proteins (identified from band position in Kurland's nomenclature as 12a and 15a) are altered significantly in mobility. It is probable that the two missing bands have new mobilities coincident with other bands in the pattern. In fact the fifth band from the bottom is noticeably intensified. However, it cannot be determined if the alteration of mobility is a reflection of the loss of function. It is expected that purification of the ribosomal proteins now in progress will answer this question EQUIVALENTS OF SPLIT OR CORE PROTEINS FzouB]~ 6. Ribosomal reconstitution of 16 S rrna from untreated ribosomes, 30 S protein from tetranitromethane inactivated ribosomes and varying amounts of either core --O-- or split proteins from untreated ribosomes. Following reconstitution, the ribosomes were collected by centrifugation and assayed for their ability to bind 14C-Phe-tRNA. The 100% level of activity was determined by assaying a reconstitution mixture containing two equivalents of both core and split proteins. Core and split proteins were prepared by the method of Traub et al. (1967) and reconstitution was by the method of Traub and Nomura (1969). amount of activity return was observed in the split protein extract. Thus the TNM must be inactivating some proteins other than the two identified in the split fraction by Traub et al. Polyacrylamide gel electrophoresis. One trivial explanation of the ability of TNM to inactivate ribosomes is that the nitration results in a loosening of the ribosome structure, ultimately leading to a dissociation of one or more essential proteins. Were this in fact the case, the site of nitration resulting in loss of activity would represent a protein not in the actual trna-poly U binding site, but rather a protein responsible for the binding of an essential protein. To examine this possibility, ribosomal proteins, extracted from TNM inactivated ribosomes, which had been subsequently pelleted by centrifugation, were analyzed by polyacrylamide gel electrophoresis (Leboy et al., 1964). Gel patterns are shown in Fig. 7. The TNM inactivated product is shown on the left and a control pattern on the right. All major bands appear to be present in the TNM treated extract except for := ==:ii: (- FIGURE 7. The electrophoretic pattern of tetranitromethane inactivated 30 S ribosomal proteins. 30 S ribosomes were treated with a 622-fold excess of TNM for one hour at 28~ Following the reaction, the ribosomes were pelleted by centrifugation and the proteins analyzed electrophoretically on polyacrylamide gels by the methods of Leboy et al. (1964). It can be seen that the control gel (right) contains two bands that are missing in the TN1V[ treated sample (left).

6 134 CRAVEN, GAVIN, AND FANNING ~SOLATION AND CHARACTERIZATION OF A MUTANT RESISTANT TO TETRACYCLINE Isolation of a tetracycline resistant mutant. A strain of E. coli was isolated which exhibits an unusually high rate of spontaneous mutation (Boettiger and Craven, in prep.). This strain shows.many interesting and useful properties. The one of most immediate concern is the appearance of mutant colonies when plated on high concentrations of tetracycline. This strain can produce large numbers of mutants resistant to at least 50/~g/ml tetracycline by direct plating. Since other workers (Reeve, 1968; Laskin and Chan, 1964) have reported that mutants resistant to low levels of tetracycline appear to be permeability resistant, we decided to try to exploit this mutator strain to obtain a tetracycline resistant mutant containing resistant protein synthetic machinery. The rationale behind our approach was based on general experiences reported with other antibiotics such as streptomycin and spectinomycin. We refer here to the observation that mutants resistant to very high levels of a given antibiotic, whose site of action is protein synthesis, tend to be resistant in vitro; whereas low level resistance tends to be located elsewhere (Mitchison, 1953). Thus we selected for mutants resistant to progressively higher concentrations of tetracycline until we reached a point where we could no longer easily go higher. The anticipation was that, having once obtained a mutant resistant to such high levels, the chances are much greater that it will be resistant in vitro. The actual sequence of mutant selection was from 50 #g/ml to 75 tug/ml, to 135 #g/ml to 150 #g/ml, and finally to 250/~g/ml tetracycline. The mutants resistant to 250 #g/ml could not be readily selected to higher quantities of the antibiotic. Resistance in vitro for polyphenylalanine formation. When the 250 #g/ml resistant strain was examined for in vitro resistance, the results presented in Table 1 were obtained. This table gives per cent inhibitions obtained at several levels of tetracycline (Tc) using the poly U directed synthesis of polyphenylalanine. The synthesizing system was developed essentially as described by Nirenberg (1964). The results given in Table 1 are typical of many experiments which have invariably shown a clear difference between the resistant strain and a sensitive control. We are therefore encouraged to suggest that this mutant contains some feature of its protein synthetic machinery which is resistant to tetracycline. Co-transduction with AroE The 250#g/ml resistant strain has two inherent disadvantages for study. The first is that it contains a number of TABLE I. THE EFFECT OF TETRACYCLINE ON POLY U DIRECTED INCORPORATION OF 14C-PHENYLALANINE INTO PROTEIN BY S-30 EXTRACTS DERIVED FROM Tc-S and Tc-R MUTANTS OF E. cell Source of S-30 Tc cone. /~g/ml Per cent inhibition To-R 4 3 Te-S 4 41 To-R l0 10 Te-S l0 50 Tc-R Tc-S Tc-R Tc-S The S-30 extracts and protein synthesizing mixtures were prepared according to methods described by Nirenberg (1964). Each reaction mixture contained 50 #c//~mole *4C-phenylalanine and 11 A~e 0 units of dialyzed S-30 fraction. Incubation time was 25 Din. Te-S, tetracycline sensitive; Te-R, tetracycline resistant. individual mutations to tetracycline resistance. The second and more serious disadvantage is that the strain is still genetically unstable. To overcome these difficulties and at the same time to learn something about the chromosomal location of these markers, we attempted to transduce the tetracycline resistance into a genetically stable background. Thus the generalized transducing phage P1 was grown on the Tc-R 25~ strain and the resultant stock infected into a strain of E. cell maintaining the AroE- marker. AroE + transductants were picked and screened for tetracycline resistance at a level of 10/zg/ml. Roughly 60% of the AroE + transductants also were able to grow on 10 jug/ml tetracycline. One of these was selected for in vitro studies. Figure 8 shows the kinetics of phenylalanine incorporation using ribosomes and supernatant derived from the tetracycline resistant transduetant and a control transductant which was tetracycline sensitive. In the absence of the antibiotic, the rate and degree of incorporation are very nearly identical for the two transductants. However, in the presence of intermediate amounts of tetracycline, the extract from the resistant transductant shows a significantly greater extent of polyphenylalanine formation. That this relationship between sensitive and resistant transductants holds over all concentrations of tetracycline is shown in Fig. 9. The experiments summarized by this figure involve measurements of polyphenylalanine formation after 25 Din of incubation over a wide range of tetracycline concentrations. Other experiments have demonstrated that this difference is maintained for even shorter incubation periods. Localization of resistance in the ribosomes. The location of the tetracycline resistance was examined by mixing supernatants with ribosomes from sensitive and resistant strains. These results are

7 BINDING SITE OF THE 30 S RIBOSOME 135 O~ O~..d..J I0 ~ 4 kl,e d 2 I00,~ Tc-R?.rc-s o ~ /o /s zo ~ T/ME (rain) FIGURE 8. The kinetics of polyphenylalanine incorporation by ribosomes and supernatant derived from tetracycline-sensitive and tetracycline-resistant transductants. Ribosomes were prepared by centrifuging an S-30 extract at 105,000 g for 2 hr. The ribosomal pellet was resuspended in buffer containing 0.01 M Tris ph 7.8, M magnesium acetate, and 0.06 M KCl. The supernatant fraction was treated with protamine sulfate (Wood and Berg, 1962). Each reaction mixture contained 0.04 M Tris ph 7.8, 0.05 M KC1, ~ MgSO4, 0.1 mm ATP, 0.25 mm GTP, M /~-mereaptoethanol, 5 A~s 0 units ribosomes, 10 [d supernatant, 10rag polyuridylic acid, 4A~ o units trna and 14C-phenylalanine (50/~e//~mole). Reaction mixtures were incubated for 25 rain at 37~ Reaction was stopped by adding 2 ml 10% TCA. The mixture was heated for 10 rain at 90~ and the resulting precipitate collected on a millipore filter. Radioactivity was counted in a Beckman liquid scintillation counter. 90 Tc-S TABLE 2. RIBOSO~&L LOCALIZATION OF TETRACYCLINE RESISTANCE IN E. C0~i TRANSDUCTANTS Per cent Ribosomes Supernatant Te Count/rain inhibition Tc-S sens Tc-S sens -t Tc-S res Te-S res ~ Tc-R sens Te-R sens ~ Te-R res Te-R res presented in Table 2 and it is apparent that polyphenylalanine formation is more sensitive to tetracycline inhibition when utilizing ribosomes derived from the sensitive transductant. Thus we can conclude that the resistance to tetracycline resides in the ribosomal fraction. Reversal of resistance by salt washing. Resistance to the antibiotic can be readily removed or inhibited by washing of the ribosomes with (NH4)2SO 4 according to the procedure published by Kurland (1966). This phenomenon is documented in Fig. 10, where it is.apparent that salt-washed resistant ribosomes have become equally as sensitive as the control preparation. Experiments thus far attempting to recover resistance by the readdition of the salt-wash supernatant have been unsuccessful. Therefore it is premature to conclude whether the resistance observed in these to :r 80 ~ R 70 9 I-,. ~" 6o z 50 I-- 2 h, 40 o I1: Ul r 30 mkia. s t HTc-s 20 I0 0 I TETRACYCLINE CONCENTRA TION ~g/ml) FIGURE 9. The effect of increasing concentrations of tetracycline on Poly U-directed polyphenylalanine synthesis. Reaction mixtures were the same as those described in Figure IO 20 so TETRACYCLINE CONCENTRATIONug~I FIGURE 10. The effect of increasing concentrations of tetracycline on poly U-directed polyphenylalanine synthesis. Reaction mixtures were the same as those described in Figure 8, except that ribosomes were fractionated with (NH4)=SO 4 according to the methods of Kurland (1966).

8 136 CRAVEN, GAVIN, AND FANNING investigations actually is removed by salt treatment or merely rendered inactive. In any event, it is anticipated that purification of this factor will be possible and will shed considerable light on the mode of tetracycline action. DISCUSSION At this juncture it seems reasonable that the protein modification reagent, tetranitromethane, will be a valuable probe of the 30 S ribosome's site of messenger and trna binding. The ultimate identification of the protein(s) responsible for loss in activity subsequent to nitration will be an important addition to the catalog of ribosomal protein function. It will be of special interest to compare the protein(s) with the streptomycin and spectinomycin proteins whose actions apparently serve to modify the function of the protein(s) directly involved in the binding. As mentioned earlier, the results of Nomura et al. presented here suggest strongly that all or most of the proteins in the ribosome modify or affect the overall ability of the ribosome to function. Thus, even with the evidence that messenger and trna can protect the binding activity from the attack by TNM, we cannot definitively conclude that we are directly nitrating the site in question. It could be that some dramatic allosteric transition is required for activity, and TNM manages to nitrate a protein important for such a process. It is difficult to rule out such a possibility, although the general characteristics of the inactivation tend to convince us that we are directly modifying the ribosomal binding site for either messenger, trna, or both. We have performed experiments with TNM inactivated ribosomes to determine if messenger binding activity has been lost, but the results are equivocal. Completely inactive ribosomes can still bind poly U as determined by sucrose gradients, but the degree of binding is roughly 15 to 20% of normal, fully active preparations. One significant conclusion the studies with TNM allow us to make is that, if we are derivatizing the actual trna binding site, then trna can interact with this site specifically even in the absence of messenger. This idea is implied by the observation that protection from inactivation occurs with trna alone. Furthermore, one can draw a similar conclusion regarding the messenger and ribosome interaction. Regarding the results with tetracycline, we can conclude that we have isolated a tetracycline resistant mutant containing ribosomes resistant in vitro. Furthermore, the mutationally altered factor appears to be located on the E. cell chromosome in a general region associated with the ribosomal proteins (more precise mapping studies are underway). However, we cannot yet conclude if this resistance is located in a ribosomal protein. This must await the final purification. ACKNOWLEDGMENTS This work was supported by funds from the National Institutes of Health (GM 15422). Thomas Fanning is a predoctoral trainee. REFERENCES DOYLE, R. J., J. BELLe, and O. A. ROHOLT Probable protein erosslinking with tetranitromethane. Biochim. Biophys. Acta 160: 274. ELLMAN, G Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82: 70. FOGEL, S., and P. S. SYPHERD Chemical basis for heterogeneity of ribosomal proteins. Prec. Nat. Acad. Sci. 59: HARDY, S. J. S., C. G. KURLAND, P. VOYNOW, and G. MORA The ribosomal proteins of E. coli I. Purification of the 30 S proteins. Biochemistry 8: KALTSCHMIDT, E., M. DZIONAYA, D. DONNER, and H. G. WITTMANN Ribosomal proteins. I. Isolation, amino acid composition, molecular weights and peptide mapping of proteins from E. coli ribosomes. Mol. Gen. Genet. 100: 364. KURLAND, C. 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9 BINDING SITE OF THE 30 S RIBOSOME ~7. Structure and function of E. coli ribosomes. IV. Isolation and characterization of functionally active ribosomal proteins. Prec. Nat. Acv~l. Sci. 58: TRAus, P., and M. NOMU~A Structure and function of Escherichia coli ribosomes. VI. Mechanism of assembly of 30 S ribosomes studies in vitro. J. Mol. Biol. 40: 391. TRAUT, R. R., and A. L. HA~L The effect of sulphydryl reagents on ribosome activity. Europ. J. Biochem. 2: 64. WOOD, W. B., and P. BEno Effect of enzymatically synthesized ribonucleic acid on amino acid incorporation by a soluble protein-ribosome system from Eacherichia coli. Prec. Nat. Acad. Sei. 48: 94.

10 The Transfer RNA Binding Site of the 30 S Ribosome and the Site of Tetracycline Inhibition Gary R. Craven, Ray Gavin and Thomas Fanning Cold Spring Harb Symp Quant Biol : Access the most recent version at doi: /sqb References This article cites 19 articles, 6 of which can be accessed free at: Creative Commons License Alerting Service Receive free alerts when new articles cite this article - sign up in the box at the top right corner of the article or click here. To subscribe to Cold Spring Harbor Symposia on Quantitative Biology go to: