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Decoding at the ribosomal A site: antibiotics, misreading and energy of aminoacyl-tRNA binding
Biochimie, 69 (1987) 803 - 813 © Soci6t~ de Chimie biologique/Elsevier, Paris
803
Decoding at the ribosomal A site: antibiotics, misreading and energy of aminoacyl-tRNA binding Horst HORNIG l, Paul WOOLLEY 2. and Reinhard L U H R M A N N l Max-Planck-Institut f a r molekulare Genetik, Ihnestrafle 63, D-IO00 Berlin 33, F . R . G . , and 2 Kemisk Institut, A a r h u s Universitet, DK-8000 ,~rhus C, D e n m a r k (Received 30-3-1987, accepted after revision 10-6-1987)
Summary - The binding of Phe-tRNA phe at the programmed ribosomal A site has been investigated using antibiotics that influence this binding in different ways. The adhesion of Phe-tRNA phe, the consumption of GTP and the extent of the peptidyl transfer reaction were monitored. All of the five known misreading-inducing antibiotics that were tested stabilised the binding of Phe-tRNA Phe after its affixture to the A site by EF-Tu with GTP hydrolysis. The stabilisation was sufficient to overcome a single mismatch in the c o d o n - a n t i c o d o n interaction. Combinations of stabilising and destabilising influences were found to be additive, thus supporting the concepts: (1) that there is a 'correct' binding energy for aminoacyl tRNA in the A site, whose reduction hampers polypeptide synthesis and whose increase makes it inaccurate by by-passing proofreading; and (2) that the different antibiotics affect the bound aminoacyl tRNA at different points. ribosome / A site I prote~n biosynthesis I misreading I antibiotic R~sum~ - D ~ c o d a g e au site ribosomal A : les antibiotiques, erreur de lecture et ~nergie de l'attachement a m i n o a c y l - t A R N . L'attachement de P h e - t A R N phe au site ribosomal A programm6 a 6t6 6tudi6 ~?1 Ullll5£llll U~'5"UHllOlOllqU~5" q u t irlJlli~'rlC'~Hl t.-~l UllUC'lrl~tll~gll U~ u t j j e r c n t e ~ ,,,u,,i~,e3. l,ou~" . . . . . .uvt,,,a . . . . . . ~,,,v,"'" .
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l'adhdrence du P h e - t A R N Phe la consommation de G T P et l'6tendue de la rdaction de transfert du peptidyl. Les cinq antibiotiques connus p o u r induire une erreur de lecture, que nous avons testds, stabilisent l'attachement du P h e - t A R N pne apr~s sa fixation au site A par E F - T u avec une hydrolyse du GTP. La stabilisation s'est rdvdl6e suffisante p o u r maftriser une erreur d'assortiment dans la rdaction codon-anticodon. Les combinaisons d'influences stabilisantes et ddstabilisantes se sont rdvdldes ~tre cumulatives, dtayant ainsi la conception scion laquelle : (1) il y a une dnergie d'attachement ~ correcte~ p o u r l'aminoacyl-tARN au site A , dont la rdduction gdne la synth~se des polypeptides et dont l'augmentation la rend infidkle en 6vitant la lecture d'dpreuves; et (2) les diffdrents antibiotiques affectent l'aminoacyl-tARN li~ d diff~rents points. ribosome I site A I biosynth~se protdique I misreading I antibiotique
Introduction In spite of continued debate on the number, identity and function of the various tRNA-binding sites on the ribosome, there remains general agreement
on the existence of an A (decoding) site, which binds the ternary complex ( E F - T u - G T P aa-tRNA) and provides a messenger-dependent discrimination between aa-tRNAs that are cognate or non-cognate to the codon that makes up part of
* Author to whom correspondence should be addressed. Abbreviations: EF-Tu: elongationfactor Tu; aa-tRNA: aminoacyltransfer RNA; 30S, 50S: 30Sand 50S ribosomal subunits;GDPNP: guanosine-5'-(fl,y-imido)triphosphate; Tris: Tris(hydroxymethyl)aminomethane.
804
H. Hornig et al.
the A site. After hydrolysis of GTP and expulsion of the (EF-Tu-GDP) complex, the aa-tRNA molecule is traditionally supposed to remain on the ribosome and subsequently to take part in peptidyl transfer, translocation and the remaining steps of the elongation cycle. It has long been known that the c o d o n anticodon interaction prior to the hydrolysis of GTP does not provide sufficient discrimination to ensure that translation takes place with sufficient accuracy at the high working speed required [1,2], and the term 'proofreading' is used to cover the various mechanisms for multiple-checking that have been postulated both for the translation of RNA into protein and for other biological copying processes. One way in which proofreading can occur in the translation process is the allowance of the possibility that aa-tRNA may dissociate from the ribosome, after GTP hydrolysis, with a dissociation rate that is higher for aa-tRNAs that are not cognate to the codon, that is, whose energy of interaction with the A site is low, since the discrimination lies in the off-rate [3, 4]. Roughly speaking, we can say that proofreading of A site-bound aa-tRNA occurs if the following inequality holds: rate (dissociation if non-cognate)>rate (next step towards peptidyl transferase)> rate (dissociation if cognate). This concept has been refined and investigated thoroughly in the past decade, and the theoretical and experimental contributions that have been made in this field are reviewed in depth l i t [,,31, t ~ - ' ~ | d J L l ~
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Antibiotics that act on the ribosome have frequently been used for the investigation of individual steps of protein biosynthesis. Numerous antibiotics are known that interfere with the binding of the ternary complex ( a a - t R N A - E F - T u - G T P ) in the elongation cycle, including many that induce misreading [6-8]. Work by Davies, Gorini and others [6, 9, 10] suggested that these antibiotics induce misreading by causing structural changes at the A site. Spirin [11] and Suzuki et al. [12] suggested that they might function by stabilising the 30S-50S interaction. However, this hypothesis was refuted by the discovery [13] of a streptomycin analo~e that does stabilise this interaction but does not induce misreading. Investigations by Ninio [4] and Gavrilova et al. [14] lent weight to the interpretation that the misreading-inducing aminoglycosides raise the speed and thus the error of protein biosynthesis. Kurland [15] emphasized the possibility that these antibiotics might affect not the site of codon-anticodon contact, but some other element of tRNA-ribosome interaction at the A site.
In earlier work [16-18], we investigated the effects of various kinds of perturbation on the prokaryotic (E. coil) ribosomal complex (70SAUGUUU-tRNAMet-Phe-tRNAPhe). The complex was prepared by the enzymic route (i.e., the A site was occupied by Phe-tRNA Phe with the help of EF-Tu and GTP), and the perturbations applied were" (1) shortening or lengthening of the UUU codon; (2)interruption of the hexanucleotide messenger, using, e.g., AUG and UUU separately; (3) replacement of one of the bases in the UUU triplet by a different base; and (4) addition of an antibiotic, viomycin (which strengthens binding of aa-tRNA in the A site) or sparsomycin (which weakens it). The behaviour of the complex in all these situations was explicable by the hypothesis that the fate of the bound aa-tRNA, after hydrolysis of GTP and before peptidyl transfer, is determined principally by the energy of binding of the aa-tRNA to the A site. This energy could be decreased by a shortened or mutated codon, an interruption of the messenger (promoting dissociation) or a destabilising antibiotic. The behaviour of the 70S complex with occupied A site was observed, using three experimental criteria" (1)the stability of the A site binding, (2) the turnover of GTP, and (3) the amount of dipeptide formed when the P site contained fMettRNA Met instead of uncharged tRNA Met. It was found that a small decrease in binding energy did not greatly reduce the ability of (EFT u - ~ l r - r n e - t R N A Phe) to bind to the A site and undergo GTP hydrolysis, but it resulted in the PhetRNA abe dissociating again and vacating the A site. Thus, while the normal binding energy resulted in stoichiometric binding of Phe-tRNA ehe to the ribosome and a low GTP turnover, binding weakened, e.g., by a single mismatching base in the codon gave a low binding level of Phe-tRNA ehe and a greatly increased GTP turnover. Dipeptide formation was rapid and quantitative under conditions of normal binding, and with weakened binding the amount of dipeptide was small, and rose slowly and linearly, as would be expected from the recycling of Phe-tRNA abe. Further weakening of the interaction (e.g., two codon bases missing) resulted in zero binding of Phe-tRNA Phe and a low or zero GTP turnover. The effect of one mismatching base in the codon could be simulated by the antibiotic sparsomycin, and it could be reversed by viomycin. Thus the response of A site-bound aa-tRNA was, empirically, largely reducible to a single parameter" the energy of the interaction between the aa-tRNA and the A site before and after GTP hydrolysis.
Antibiotics and misreading at the ribosomal A site Detailed effects, such as those observed with the non-cognate codon CUU, which gives a weak but non-productive specific stabilisation of PhetRNA Phe in the A site [18], do not affect qualitatively the general conclusion. The effects of perturbation of aa-tRNA in the A site could thus, in the first instance, be reduced to increments and decrements of its binding energy. If the binding energy is a variable dependent upon the sum of many, approximately independent interactions, then it should be possible to extend the range of stabilising and destabilising factors and to see to what extent they can be made to reinforce one another or to cancel out. The purpose of this investigation was therefore to find out how other antibiotics influence this test system, and to what extent their behaviour can also be rationalised in this simple way.
Materials and methods The isolation and purification of tight-couple 70S ribosomes from E. coli followed standard methods [19]. The preparation and characterisation of oligonucleotides, the charging of tRNA and the assay conditions for aa-tRNA binding, phosphate turnover and dipeptide formation have been described elsewhere [16-18, 20]. In summary, the assays were conducted as follows, all at 37°C. 25 pmol of 70S ribosomes, 50 pmol of deacylated tRNAMet and 2 nmol of messenger oligoribonucleotide were pre-incubated for 2 min in 75 pl of the standard h n f f e r , n ~ d in pre~ous ~tl,d;,~ ~fis/Tris hydrochloride 25 mM, pH 7.5, ammonium chloride 50 mM, 2-mercaptoethanol 2.5 mM and magnesium acetate 11 mM or, in certain cases where stated, 5 raM). If required, antibiotic was added in 5/zl of dimethyl sulphoxide and the pre-incubation was repeated. To start the reaction, 25 pl of a mixture of EF-Tu (120 pmol), GTP (1 nmol) and [3H]aa-tRNA, pre-incubated in the same buffer, was added. For the assay of aa-tRNA binding, the amount of radioactivity bound to the ribosomes was determined by a Millipore filter test. For dipeptide synthesis, deacylated t R N A Met in the above test was replaced by fMet-tRNAfMet and the amount of radioactive dipeptide formed was measured by extraction into ethyl acetate. For the assay of GTP hydrolysis, [7'-32p]GTP (0.5 Ci/mmol), phosphoenol pyruvate (2 mM) and pyruvate kinase (0.1 mg/ml) were also included, the reaction was stopped with perchloric acid and the inorganic phosphate liberated was determined by extraction with molybdate into isopropyl acetate. Antibiotics were purchased from Sigma (Munich); several were kindly donated by Dr. E Cundiiffe (Leicester University). The concentrations of antibiotics in the assays were reasonably high (greater than the dissociation constants, where these are known), so the ribosomes may be considered as being saturated with antibiotic, or nearly so.
805
Results Antibiotics that induce misreading The occupation of the A site by Phe-tRNA Phe was monitored as a function of the length of the A site codon in the presence of various antibiotics. Fig. I shows typical results. On the left, the complete codon UUU gives a plateau of Phe-tRNA Phe binding and phosphate consumption (A). Weakening of the A site affinity for Phe-tRNA Phe, by use of the truncated codon UU, gives a low plateau and a high phosphate consumption, which are due to dissociation and continual recycling of PhetRNA Phe (B). Further weakening (truncated codon U) means that the residence time of the ternary complex is reduced so much that GTP hydrolysis hardly takes place (C). On the right, the stabilising effect of neomycin is seen. While the antibiotic has only a slight effect on the already stable complex with the triplet codon, the weakness of the UU codon is compensated for (E). Even the extremely weakly-binding codon U is strengthened a little, so that more GTP is hydrolysed, even though all the Phe-tRNA Phe dissociates again (F). Similar results were o b t a i n e d with the misreading-inducing aminoglycoside antibiotics streptomycin, kanamycin and gentamicin and were also indicated in an earlier study with viomycin [17]. However, the aminoglycoside antibiotic spectinomycin, which does not induce misreading, had no effect in these experiments, the results of which are summarised in Table I, columns 1-3. All the misreading-inducing antiobiotics tested provide a stabilisation of aa-tRNA in the A site that is roughly equivalent to the destabilisation caused by a missing codon base. Two missing codon bases can only be compensated for to a small extent. Although the stability of A site binding is influenced, its geometry cannot be grossly distorted, as is shown by the almost invariant yield of dipeptide (80-85°7o). The loss of one base from the codon is thus compensated for by the presence of a misreadinginducing antibiotic. The fact that the loss of two bases is not compensated, so as to appear like the loss of only one, suggests the possibility that the stabilising effect of these antibiotics may be effective only after GTP hydrolysis. However, this is not the case, as shown by repeating the experiment replacing GTP by its non-hydrolysable analogue GDPNP. It is already known that when cognate ternary complex with GDPNP is offered to the A site, initial binding takes place, but the EF-Tu is not discharged from the 70S complex, and the
806
H. Hornig et al.
aminoacylated 3' end of the tRNA does not take part in the peptidyl transferase reaction [21]. The effect of replacing GTP with GDPNP is shown in Table I (4th column): the stabilisation of A site-bound Phe-tRNA Phe by misreadinginducing antibiotics also occurs when the Phe-
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tRNA Phe is bound to the A site in its pre-GTPhydrolysis conformation, along with EF-Tu. In the presence of the non-hydrolysable GTP analogue, none of the Phe-tRNA Phe is able to adopt its postGTP-hydrolysis conformation, as shown by the absence of dipeptide formation, found for all an-
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Fig. 1. P site complexes were prepared by incubation of 70S ribosomes and tRNA~ et, with the oligoribonucleotide AUG(U)n fis messenger. At time zero, ternary complex (EF-Tu-GTP-Phe-tRNAPhe) was added and the course of Phe-tRNA Phe binding and GTP hydrolysis was monitored. A : messenger = A U G U U U ; B: A U G U U ; C : A U G U . D - F , same as A - C , but 100/zM neomycin was added to the pre-incubation mixture (see Materials and Methods).
Antibiotics and misreading at the ribosomal A site tibiotics (the 1°70 observed is not significant). Again, neomycin is able to compensate for the loss of one base in the codon (Table I, 5th column). In all these experiments, spectinomycin has no stabilising or destabilising effect, which confirms that the stabilisation by these antibiotics is indeed correlated with their ability to induce misreading. The same effect is observed when, instead of a s h o r t e n e d c o d o n , a n o n - c o g n a t e c o d o n is employed. A single base substitution has an effect roughly equal to that of a single deletion, and, as Table II shows, this decrease in binding affinity can be made good by the addition of neomycin. Again,
807
there is no impairment of the ability of Phet R N A Phe, once bound, to form dipeptide. In Table II, the stoichiometric ratio of G T P hydrolysed to P h e - t R N A Phe bound should be noted. For stable binding, fewer than 2 G T P molecules are hydrolysed per Phe-tRNAl'he: for unstable binding, this ratio exceeds 30 during the 3 min incubation and continues to rise thereafter. Another way of weakening the A site binding of a a - t R N A is to interrupt the hexanucleotide messenger, by using for example (AUG + UUU) or ( A U G U + UU) [16]. Although this weakening has no counterpart in vivo, its effects are of con-
Table I. Yields of bound Phe-tRNA l'he and dipeptide for various antibiotics, for normal and shortened A site codons and for GTP/GDPNP.
Antibiotic
AUGUUU, GTP
AUGUU, GTP
AUGU, GTP
AUGUUU, GDPNP
AUGUU, GDPNP
None Neomycin Gentamicin Kanamycin Streptomycin Spectinomycin
68 (85) 74 (85) 74 72 72 68
8 72 70 68 68 8
4 (< 1) 7 (< 1) 7 6 6 4
20 76 74 74 70 20
4 12 4
(> 100)a (84) (81) (83) (81)
(1) (1) (1) (1) (1) (1)
70S ribosomes (25 pmol) were programmed at the P site by incubation with messenger and tRNA~et (for the A site binding assay) or fMet-tRNA~et (for the dipeptide assay). After addition of 100 tzM antibiotic, if appropriate, the required ternary complex (EFTu and Phe-tRNATM pre-incubated together with GTP or GDPNP) was added. After reaction to completion (3 min; in some eases 10 min was allowed) the binding or dipeptide measurement was made [16]. The yield of bound Phe-tRNApheis stated as a percentage of the number of ribosomes (maximum obtainable = 75070). The yield of dipeptide, where measured, is stated in parentheses as a percentage of the amount of bound Phe-tRNAPh% a The percentage dipeptide yield has no meaning this case, since, in accordance with the steady-state binding pattern, weak A site occupation results in a slow, linear accumulation of dipeptide, to give a 'yield' which increases steadily and, at length, exceeds 100070 w.r.t, bound Phe-tRNAPhe.
Table II. Yields of bound Phe-tRNA Phe and dipeptide for various antibodies and altered A site codons.
Oligonucleotide
AUGUUU AUGCUU AUGUU AUGUCU AUGUUG AUGGUU AUG + UUU AUG + UUU AUG+UUU AUG + UUU
No antibiotic
+ Antibiotic
Antibiotic
Yield
GTP hydrolysed
Yield
GTP hydrolysed
68 (85) 22 8 7 6 4 1.6 1.6 1.6 1.6
128 108 260 252 256 108 <2 <2 <2 <2
74 (85) 66 (78) 65 (84) 56 (80) 62 (83) 48 (76) 40 (78) 0.6 (= 70) 0.6 (=80) 0.4 (= 75)
120 115 120 153 144 110 120 160 160 <5
neomycin neomycin neomycin neomycin neomycin neomycin neomycin gentamicin kanamycin streptomycin
The neomycin concentration was 100 btM. Yield of bound Phe-tRNAPheand turnover of GTP are given as aT0of ribosomes present; dipeptide yield, where measured, is given in parentheses as 070of bound Phe-tRNAphe.
H. Hornig et al.
S08
tion, so results are presented both for 11 mM and 5 mM Mg 2+ (Table III, 1st and 2nd columns). Four of them showed no inhibitory effect and were not investigated further. The remaining nine were divided into categories, according to their ability to suppress as well the binding of the tertiary complex ( E F - T u GDPNP-Phe-tRNAPhe), which contains the nonhydrolysable analogue of GTP. Only two of them, chlortetracycline and thiostrepton, suppressed its binding. The remaining seven are described in the literature as peptidyl transferase inhibitors, and our measurements of dipeptide formation (Table III) agree with this. Yet they clearly have a further influence, as shown by an earlier experiment [17] with sparsomycin, whose results we recapitulate briefly. If the time course of Phe-tRNA Phe binding and GTP hydrolysis are measured as in Fig. 1, then the following pattern emerges. (1) For the messenger AUGUUU, sparsomycin lowers the plateau level of bound Phe-tRNA Phe from 0.65 mol/ribosome (Fig. 1A) to about 0.17 mol/ribosome (Fig. 2D). However, it remains a clear plateau. In contrast, the phosphate turnover is more than doubled and
siderable interest. Like the other kinds of destabilisation (above), this weakening was compensated for by neomycin, an effect which we have already observed for viomycin [16]. Interestingly, gentamicin and kanamycin have a weaker influence and streptomycin does not appear to work at all (Table II). There is already a hint (Table II) that neomycin exerts a stronger stabilising effect, and streptomycin a weaker one, than do the others. Apparently the interruption of the messenger has a greater destabilising influence and thus calls for a greater stabilisation, so that it discriminates more strongly between the degrees of stabilisation offered by the different antibiotics. The stronger discrimination may also be due to a different site of action, e.g., with the site of interaction of neomycin being closest to the anticodon.
Antibiotics that inhibit peptidyl transfer Thirteen antibiotics frequently described as being A site active were tested for their ability to inhibit the binding of Phe-tRNA abe to the cognately programmed A site. It was observed that some had a greater effect at lower magnesium ion concentra-
Table HI. Inhibition of binding of Phe-tRNA phe in the A site programmed by A U G U U U , in the presence of GTP or G D P N P . A ~.=I.=~:~ J"kJ[l LI UIU
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11 mM Mg 2+
5 mM Mg 2+
None
80
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38
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63
Chloramphenicol Streptogramin Sparsomycin Niddamycin Carbomycin Clindamycin Lincomycin
35 20 28 31 30 61 62
( = 1) ( --- 1) ( = 1) ( = 1) ( = 1) ( = 1) ( = 1)
15 4 11 12 13 14 15
( = l) ( = 1) ( = 1) (=1) (=1) (--- l)
62 64 63 65 62 64 61
Chlortetracycline Thiostrepton
27 72
3 9
Erythromycin Thylosin Spiramycin Bottromycin
77 80 77 79
38 39 39 37
(----l)
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I l t ~ l U bound r ' n e - t ~ ' ~ with G D P N P (5 mM Mg 2+)
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Reactions were carried out at two Mg2+ concentrations (see text). Concentrations of antibiotics were: chlortetracycline: 150/zM; thiostrepton: 21/zM ; streptogramin and sparsomycin: 55/zM; others: 100/zM. Incubation time was 3 min. Yield of bound PhetRNA phe is given as o70 of ribosomes present: dipeptide yield, where measured, is stated in parentheses as % of bound Phe-tRNA phc. The messenger was AUGUUU for the experiments with tertiary complex including GTP and poly(U) with tRNA phe in the P site for those including GDPNP.
Antibiotics and misreading at the ribosomal A site
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Time (rain) Fig. 2. Time course of Phe-tRNA Phe binding and GTP hydrolysis in the presence of the messenger AUGUUU (cf. Fig. IA). A: no antibiotic; B: plus 150/zM chlortetracycline; C: plus 21 btM thiostrepton; D: plus 55 /zM sparsomycin [17]. Measurements A - C were made at a lower Mg 2+ concentration (see text), which depresses all binding levels.
does not reach a plateau, but continues to rise. (2) For the messenger AUGUU, which gives a very low steady-state binding level of Phe-tRNA Phe and a high, rising GTP turnover (Fig. IB), the addition of sparsomycin has no effect (not illustrated). Both of these observations indicate that the inhibiting effect of sparsomycin at the peptidyl transferase site is accompanied by a physical (presumably steric) blockage that also interferes with, and destabilises, the binding of the aminoacylated CCA end of the A site-bound tRNA. This has less destabilising effect on the A site aa-tRNA molecules than, for example, a noncognate codon (compare Fig. 1B and 2D), but it nevertheless leads to an elevated rate of dissociation of aa-tRNA from the A site and thus both to a lower stationary-state binding plateau and to a higher, continuing consumption of GTP.
Antibiotics that block binding o f the ternary complex Of all the antibiotics tested, only two, tetracycline and thiostrepton, were found to depress the bind-
809
ing level of cognate ternary complex containing the non-hydrolysable GTP analogue (Table lII). Both of these are known to be A site inhibitors [22], but there have been contradictory ideas about their mechanism of action [23-25] and, in the case of tetracycline, its binding site (reviewed in [26]). We therefore subjected them to closer investigation. They both inhibited Phe-tRNA ehe binding more efficiently at a lower magnesium concentration, so most measurements are made both at standard (11 raM) and low (5 raM) magnesium concentrations. Although these inhibitors weaken the interaction between the A site and Phe-tRNA vhe, it is clear that they do this in a way different from that of mismatching bases and other inhibitors such as sparsomycin (Table IV and Fig. 2). This is because a small destabilisation leads not to a higher, but to a lower phosphate consumption, which does not proceed indefinitely but rapidly reaches a plateau, at a level depressed in proportion to the depression of the Phe-tRNA Phe binding level. Another difference between these two antibiotics and sparsomycin is their effect on the behaviour of A sites programmed with an incomplete triplet (messenger: AUGUU, cf. Fig. 1B). As mentioned, sparsomycin has virtually no effect on this system. However, if thiostrepton is included in the experiment described in Fig. IB, then the phosphate turnover drops to 25070, and to < 5°70 in the presence of chlortetracycline. This is clearly consistent with the observation (Table III) that these antibiotics inhibit the approach of ternary complex, since this naturally results in a drop in the amount of GTP hydrolysed. This drop is not due to inhibition of the actual GTP hydrolysis, since this GTPase activity is not blocked by thiostrepton [27].
Effects o f simultaneously-bound antibiotics If the concept of incremental stabilisation and destabilisation of Phe-tRNA Phe in the A site is correct, then it follows as a corollary that different antibiotics, acting simultaneously at different points on the A site complex, should tend to reinforce each other or to cancel each other out. With the large numbers of antibiotics tested, this suggests many possible combinations with which to experiment. However, in order to test the concept, we restricted ,this investigation to combinations of one representative from each group. These were: neomycin, for the antibiotics that induce misreading and stabilise aa-tRNA in the A site; streptogramin, for the antibiotics that inhibit the peptidyl transferase centre and destabilise the 3' terminus; and chlortetracycline, for the antibiotics that interfere with
H. Hornig et al.
810
the binding of the ternary complex to the A site prior to GTP hydrolysis. The effects of combining neomycin and streptogramin are shown in Table V (upper half). As expected, streptogramin suppresses and neomycin stimulates the binding of Phe-tRNA Phe to the A site. When both are present, either with AUGUUU or with the truncated AUGUU as messenger, the combined binding effect is largely the same as that seen when neither is present. However, the presence of streptogramin also abolishes the peptidyl transferase reaction.
T a b l e I V . Y i e l d o f b o u n d P h e - t R N A Phe,
active against ternary complex Yield
Antibiotic
stimulation of GTP hydrolysis and dipeptide yield in the presence of antibiotics
binding.
of bound
11 m M
None Thiostrepton Chlortetracycline
The combination of chlortetracycline and neomycin gave similar results (Table V, lower half). This is of greater significance, because it shows that the stabilisation by neomycin operates on aa-tRNA in the A site not only after hydrolysis of CTP, but also before. Further, it was shown that spectinomycin had no effect in conjunction with chlortetracycline, just as it had none alone in these assays (Table I). This strengthens our earlier deduction that spectinomycin has a mode of action different from that of the other aminoglycosides.
Phe-tRNA
M g 2+
5 mM
80 (82)
Mol GTP
Phe M g 2+
11 m M
38 9 3
72 (80) 28 (79)
•
•
lt~ll~V~.,L~
Vl
~IIIIUILCI.I£1wUUO
lJll,,Ol,,,aal.,~,.
Va
M g 2+
5 mM
128 120 43
~aaL~.~.,JaaaoLx~.~aaj-r.,~,v.xaz~
1Jr.Laao
M g 2+
68 13 6
Bound P h e - t R N A Phe a n d G T P hydrolysis is expressed in molecules per 100 ribosomes age of bound P h e - t R N A Phe.
la~UlIK;
hydrolysed
vl
and dipeptide,
in parentheses as the percent-
r.a~xal.xvJaVl.a-~o.
Oligonucleotide
Streptogramin
Neomycin
Chlortetracycline
Yield bound
AUGUUU AUGUUU AUGUUU AUGUUU
+ +
+ +
-
80 8 84 68
(80) (1) (82) (1)
AUGUU AUGUU AUGUU AUGUU
+ +
+ +
-
8 2 80 19
(84)
AUGUUU AUGUUU AUGUUU AUGUUU
-
+ +
+ +
80 20 84 76
(83) (79) (82) (82)
AUGUU AUGUU AUGUU AUGUU
-
+ +
+ +
8 4 80 10
Phe-tRNA
(84)
Incubation was under standard conditions, 11 m M M g 2+. Antibiotic concentrations were as in T a b l e l l I . The yield of bound percentage of ribosomes present, the yield of dipeptide in parentheses as a percentage of bound spectinomycin to the binding assay with or without chlortetracycline had no effect.
t R N A Phe is expressed as a t R N A TM. The addition of
Phe
PhePhe-
Antibiotics and misreading at the ribosomal A site
Discussion Fourteen of the nineteen antibiotics studied can be classified into three groups, according to their effects on aa-tRNA bound at the ribosomal A site. I. Neomycin, viomycin, gentamicin, kanamycin and streptomycin stabilise aa-tRNA in the A site both before and after GTP hydrolysis. By doing this, they cause errors, in particular in proofreading (dissociation of non-cognate aa-tRNA after GTP hydrolysis). They appear to have a stabilising effect which diminishes in the above order, as can be seen in Table I. (The placing of viomycin is based on its ability [16] to stabilise aa-tRNA effectively in the presence of an interrupted messenger, which otherwise only neomycin was able to do; see Table II.) It is worthy of note that both neomycin and viomycin, apparently the strongest stabilisers of aa-tRNA in the A site, are regarded not only as inducers of misreading, but also as inhibitors of translocation ([28], reviewed in [29]). If they cause aa-tRNA to bind tightly to the A site, then inhibition of translocation, to some degree, is a necessary consequence. No member of this group has any influence on the peptidyl transferase centre, whose efficiency is unimpaired by their presence. II. Chlortetracycline and thiostrepton interfere with the binding of ternary complex. It is consistent with their destabilising, rather than stabilising, ,,, ,~,~-,.,,,,.~ . . t , , ~ A ~tc that neither oz these antibiotics induces misreading. For both antibiotics, GTP hydrolysis is reduced in proportion to the reduced aa-tRNA binding (Fig. 2), while the peptidyl transferase reaction is unaffected (Table IV). The latter finding has also been reported for tetracycline by others [30]. The fact that chlortetracycline and thiostrepton have the same effect is hard to interpret structurally, because the presumed sites for tetracycline and thiostrepton binding are very far apart on the ribosome. The former antibiotic binds to protein $7 (as shown by photolytic cross-linking[31]), close to a recent placing of the A site [32], but the latter binds to a region of the 23S RNA that is thought to be some 100 ~. away from this [33]. Using the fluorescence anisotropy changes associated with the immobilisation of tetracycline on binding to ribosomes [26], we have shown that the affinity of the A site for Phe-tRNA Phe is reduced by some 80070, if tetracycline is already bound to its strong binding site on the 30S subunit [34]. This applies for both enzymic and non-enzymic occupation of the A site. It thus appears that the
811
suppression of the binding plateau (without destabilisation of A site-bound Phe-tRNA Phe) in Fig. 2 is due simply to effective non-availability of A sites of ribosomes to which a tetracycline molecule is already bound. The division of the population of ribosomes by tetracycline into 'available' and 'non-available' is consistent with our observation [26] that the tetracycline-ribosome binding takes a long time to equilibrate. This argument cannot apply to thiostrepton, which has a very high constant of binding to ribosomes [35] and yet puts only = 75°7o of them out action (Fig. 2; Table IV), so that (especially in view of the structural consideration above) there may be some differences in the mechanism that our data do not reveal directly. III. Streptomycin, sparsomycin, niddamycin, carbomycin, chloramphenicol, clindamycin and lincomycin act as antibioticsby abolishing totallythe activityof the peptidyl transfcrasecentre. But they also destabilisethe aa-tRNA bound in the A site, which makes them of interestas tools for studying aa-tRNA-ribosome binding. The strongest destabifiserswere found to be streptogramin and sparsomycin, which are also the only inhibitorsof P h e - t R N A binding in the Phe-tRNA/poly(U) binding test [22,36, 37]. Of the antibioticstested,only crythromycin, bottromycin, thylosin, spiramycin and spectinomycin had no effect on A site-bound aa-tRNA. Of these, erythromycin does not inhibitpeptidyl transferper se [35], which agrees with our data if it acts without affecting the A site. Bottromycin is claimed to inhibit the binding of aa-tRNA to the A site [22], an assertion which our data throw doubt on, since no effect of bottromycin on aa-tRNA binding was observed (Table III). However, the purpose of these experiments was less to investigate the mechanisms of action of the antibiotics than to investigate the connection between classes of antibiotic and the behaviour of aa-tRNA in the A site, setting this in relation to other stabi!t~singand destabilising factors. It is also clear that t,~lelethal quality of the antibiotics is not necessarily connected with the stability of bound aa-tRNA: for example, the destabilisation by sparsomycin and others is clearly incidental to peptidyl transferase inhibition, while gentamicin has a complex spectrum of effects [22]. None the less, it is interesting that none of the nineteen antibiotics tested destabilised aa-tRNA after GTP hydrolysis only. This may suggest that the structural difference in A site tRNA before and after GTP hydrolysis is not very great. The hypothesis formulated earlier [18], that the behaviour of aa-tRNA in the A site is determined
812
H. Hornig et al.
by the requirementof 'correct' binding energy, and that various structural factors contribute energetic increments or decrements, appears to be supported. Decrements are provided by (l) incorrect binding of the aminoacylated 3' end of the tRNA due to the presence of a peptidyl transferase inhibitor; (2) a missing codon base; (3) a non-cognate codon base, where the size of the decrement depends on the 'badness' of the mismatch; (4) destabilisation of the messenger, for example by interruption of the sugar-phosphate chain, which promotes dissociation; and (5) A site-destabilising antibiotics. Increments are supplied by certain stabilising antibiotics. These conclusions are in good agreement with the results of other groups, including the observation of weakened binding of tRNA non-cognate to the A site in the presence of the messenger poly(U) [28] with accompanying stimulated GTP hydrolysis [38-41] and stabilisation by neomycin or streptomycin [42-44]. In each case, the result of what we would term a binding energy decrement (one incorrect base pair) or increment (misreadinginducing antibiotic) corresponds with that found in the present work. The energetic increments and decrements can be balanced off against one another so that they cancel out. It is particularly interesting that this should be possible for the simultaneous addition of different antibiotics, since it lends support to the picture of separate and largely independent points of action for many antibiotics. This is supported tentatively by the observation of different points of binding of the misreading-inducing antibiotics, e.g., streptomycin and neomycin [29, 45]. The pursuit of combination experiments, such as presented in Table V, may lead to more information about the mechanisms of action of various antibiotics. The fact that antibiotics with the stabilising propetty are precisely those that induce misreading provides strong conf'mnation of the hypothesis that the ability of aa-tRNA to dissociate from the A site after GTP hydrolysis, if its binding energy is lower than normal by a decrement corresponding to a single codon-anticodon mismatch, is a part of the mechanism ensuring ribosomal accuracy. The effect of losing an AU base pair in the binding of PhetRNA phe is about the same as that of losing a GC base pair in the binding of Pro-tRNA Pr° [17], confirming that not just the codon but the whole tRNA molecule has evolved so as to bring the total energy of binding of each individual tRNA species to precisely the right value [46]. There is increasing evidence that streptomycinresistant mutant ribosomes have, in vitro, a greater
missense suppression, a higher accuracy and a higher GTP turnover per peptide bond made than do wild type ribosomes (reviewed in [2]). In the context of the present work, this would suggest that the mutant has an A site with lowered affinity for all aa-tRNAs after GTP hydrolysis (perhaps also before GTP hydrolysis, but this would not necessarily affect the accuracy). It would be interesting to assay such ribosomes in vitro in the manner described here, so as to investigate the magnitude of the decrement and its (anticipated) generality for all tRNA species.
References 1 Thompson R.C. & Karim A.M. (1982) Proc. Natl. Acad. Sci. USA 79, 4922-4926 2 Ehrenberg M., Kurland C.G. & Blomberg C. (1986) in: Accuracy in Molecular Processes (Kirkwood T.B.L., Rosenberger R.F. & Galas D.J., eds.), Chapman and Hall, London, pp. 329-361 3 Hopfield J.J. (1974) Proc. Natl. Acad. Sci. USA 71, 4135-4139 4 Ninio J. (1974) J. MoL BioL 84, 297-313 5 Kirkwood T.B.L., Rosenberger R.F. & Galas D.J. (eds.) (1986) in: Accuracy in Molecular Processes, Chapman and Hall, London 6 DaviesJ., Gorini L. & Davis B.D. (1965)Mol. Pharmacol. 1, 93-106 7 Atkins J., Elsevier D. & Gorini L. (1972) Proc. Natl. Acad. Sci. USA 69, 1192-1195 8 BenvenisteR. & Davies J. (i973) Antimicrob. Agents Chemiother. 4, 402 9 DaviesJ., Gilbert W. & Gorini L. (1964)Proc. Natl. Acad. Sci. USA 51, 883-890 10 Gorini L. (1974) in: Ribosomes (Nomura M., Tissi~res A. & Lengyel P., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 791-803 11 SpirinA.S. (1972) in: Molecular Mechanisms o f Antibiotic Action on Protein Biosynthesis and Membranes (Mufioz E., Gracia-Ferrandiz F. & Vasquez
D., eds.), Elsevier, Amsterdam, pp. 11-27 12 Suzuki J., Hori M. & Llmezewa H. (1968) J. Antibiot. 21,571-573 13 Cousin M.-A., Lando D., Ojasoo T. & Raynaud J.-P. (1977) Biochimie 59, 59-63 14 GavrilovaL.P., Kostiashkina O.E., KotelianskyV.E, Rutkevitch N.M. & Spirin A.S. (1976) J. MoL Biol. 101, 537-552 15 Kurland C.G. (1977) Annu. Rev. Biochem. 46, 173-200 16 LfihrmannR. (1980) Nucleic Acids Res. 8, 5813-24 17 Hornig H., WooHeyP. & Lfihrmann R. 0983) FEBS Lett. 156, 311-315 18 Hornig H., Woolley P. & Lfihrmann R. (1984) J. Biol. Chem. 259, 5632-5636
A n t i b i o t i c s a n d misreading at the ribosomal A site
19 NoU M., Hapke B., Schreier M.H. & Noll H. (1973) J. Mol. Biol. 75, 281-294 20 Hornig H. (1984) Dissertation: Mechanismus der aa-tRNA Bindung, Technische Universit~it, Berlin 21 Kaziro Y. (1978) Biochim. Biophys. Acta 505, 95-127 22 Cundliffe E. (1980) in: Ribosomes: Structure, Function and Genetics (Chambliss G., Craven G.R., Davies J., Davis K., Kahan L. & Nomura M., eds.), University Park Press, Baltimore, pp. 555-581 23 Gordon J. (1969) J. Biol. Chem. 244, 5680-5686 24 Kinoshita T., Liou Y.-F. & Tanaka N. (1971) Biochem. Biophys. Res. Commun. 29, 356-361 25 Modolell F., Cabrer B., Parmeggiani A. & Vasquez D. (1971)Proc. Natl. Acad. Sci. USA 68, 1796-1800 26 Epe B. & Woolley P. (1984) EMBO J. 3, 121-126 27 Ballesta J.P.G. & Vasquez D. (1972) Proc. Natl. Acad. Sc~. USA 69, 3058-3062 28 Cabafias M.J. & Modolell J. (1980) Biochemistry 19, 5411-5416 29 Gale E.F., Cundliffe E., Reynolds P.E.; Richmond M.H. & Waxing M.H. (1981) in: The Molecular Basis o f Antibiotic Action, Wiley, London, pp. 402-547 30 Semenkov Y.P., Makarov E.M., Mahnko V.I. & Kirrilov S.V. (1982) FEBS Lett. 144, 125-129 31 Goldman R.A., Hasan T., Hall C.C., Strycharz W.A. & Cooperman B.S. (1983) Biochemistry 22, 359-368 32 Ofengand J., Ciesiolka J., Denman R. & Nurse K. (1986) in: Structure, Function and Genetics o f Ribosomes (Hardesty B. & Kramer G., eds.), Springer-Verlag, New York, pp. 473-494
813
33 St6ffler G. & St6ffler-Meilicke M. (1986) in: Structure, Function and Genetics o f Ribosomes (Hardesty B. & Kramer G., eds.), Springer-Verlag, New York, pp. 28-46 34 Epe B., Woolley P. & Hornig H. (1987) FEBS Lett. 213, 443-447 35 Cundliffe E., (1986) in: Structure, Function and Genetics o f Ribosomes (Hardesty B. & Kramer G., eds.), Springer-Verlag, New York, pp. 586-604 36 Ravel J.M., Shorey R.L. & Shive W. (1970) Biochemistry 9, 5028-5033 37 Chinali G., Sprinzl M., Parmeggiani A. & Cramer F. (1974) Biochemistry 13, 3001-3010 38 Thompson R.C., Dix D.B., Gerson R.B. & Karim A.M. (1981) J. Biol. Chem. 256, 81-86 39 Thompson R.C. & Stone P. 0977) Proc. Natl. Acad. Sci. USA 74, 198-202 40 Donner D., ViUems R. & Kurland C.G. (1978)Proc. Natl. Acad. Sci. USA 75, 3192-3195 41 Ruusala T., Ehrenberg M. & Kurland C.G. (1982) EMBO J., 1 741-745 42 Yates J.L. (1979) J. Biol. Chem. 254, 11550-11554 43 Thompson R.C., Dix D.B., Gerson R.B. & Karim A.M. (1981) J. Biol. Chem. 256, 6676-6681 44 Campuzano S., Cabafias M.J. & Modolell J. (1979) Eur. J. Biochem. t00, 133-139 45 Gris6-Miron L., Noreau J., Melancon P. & BrakierGingras L. (1981) Biochim. Biophys. Acta 656, 103-110 46 Ninio J. & Claverie P. (1971) J. Mol. Biol. 56, 63-82
803
Decoding at the ribosomal A site: antibiotics, misreading and energy of aminoacyl-tRNA binding Horst HORNIG l, Paul WOOLLEY 2. and Reinhard L U H R M A N N l Max-Planck-Institut f a r molekulare Genetik, Ihnestrafle 63, D-IO00 Berlin 33, F . R . G . , and 2 Kemisk Institut, A a r h u s Universitet, DK-8000 ,~rhus C, D e n m a r k (Received 30-3-1987, accepted after revision 10-6-1987)
Summary - The binding of Phe-tRNA phe at the programmed ribosomal A site has been investigated using antibiotics that influence this binding in different ways. The adhesion of Phe-tRNA phe, the consumption of GTP and the extent of the peptidyl transfer reaction were monitored. All of the five known misreading-inducing antibiotics that were tested stabilised the binding of Phe-tRNA Phe after its affixture to the A site by EF-Tu with GTP hydrolysis. The stabilisation was sufficient to overcome a single mismatch in the c o d o n - a n t i c o d o n interaction. Combinations of stabilising and destabilising influences were found to be additive, thus supporting the concepts: (1) that there is a 'correct' binding energy for aminoacyl tRNA in the A site, whose reduction hampers polypeptide synthesis and whose increase makes it inaccurate by by-passing proofreading; and (2) that the different antibiotics affect the bound aminoacyl tRNA at different points. ribosome / A site I prote~n biosynthesis I misreading I antibiotic R~sum~ - D ~ c o d a g e au site ribosomal A : les antibiotiques, erreur de lecture et ~nergie de l'attachement a m i n o a c y l - t A R N . L'attachement de P h e - t A R N phe au site ribosomal A programm6 a 6t6 6tudi6 ~?1 Ullll5£llll U~'5"UHllOlOllqU~5" q u t irlJlli~'rlC'~Hl t.-~l UllUC'lrl~tll~gll U~ u t j j e r c n t e ~ ,,,u,,i~,e3. l,ou~" . . . . . .uvt,,,a . . . . . . ~,,,v,"'" .
.
.
.
.
.
.
.z_.
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.
.
.
.
.
.
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-" . . . . . . . . . . .
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.
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l'adhdrence du P h e - t A R N Phe la consommation de G T P et l'6tendue de la rdaction de transfert du peptidyl. Les cinq antibiotiques connus p o u r induire une erreur de lecture, que nous avons testds, stabilisent l'attachement du P h e - t A R N pne apr~s sa fixation au site A par E F - T u avec une hydrolyse du GTP. La stabilisation s'est rdvdl6e suffisante p o u r maftriser une erreur d'assortiment dans la rdaction codon-anticodon. Les combinaisons d'influences stabilisantes et ddstabilisantes se sont rdvdldes ~tre cumulatives, dtayant ainsi la conception scion laquelle : (1) il y a une dnergie d'attachement ~ correcte~ p o u r l'aminoacyl-tARN au site A , dont la rdduction gdne la synth~se des polypeptides et dont l'augmentation la rend infidkle en 6vitant la lecture d'dpreuves; et (2) les diffdrents antibiotiques affectent l'aminoacyl-tARN li~ d diff~rents points. ribosome I site A I biosynth~se protdique I misreading I antibiotique
Introduction In spite of continued debate on the number, identity and function of the various tRNA-binding sites on the ribosome, there remains general agreement
on the existence of an A (decoding) site, which binds the ternary complex ( E F - T u - G T P aa-tRNA) and provides a messenger-dependent discrimination between aa-tRNAs that are cognate or non-cognate to the codon that makes up part of
* Author to whom correspondence should be addressed. Abbreviations: EF-Tu: elongationfactor Tu; aa-tRNA: aminoacyltransfer RNA; 30S, 50S: 30Sand 50S ribosomal subunits;GDPNP: guanosine-5'-(fl,y-imido)triphosphate; Tris: Tris(hydroxymethyl)aminomethane.
804
H. Hornig et al.
the A site. After hydrolysis of GTP and expulsion of the (EF-Tu-GDP) complex, the aa-tRNA molecule is traditionally supposed to remain on the ribosome and subsequently to take part in peptidyl transfer, translocation and the remaining steps of the elongation cycle. It has long been known that the c o d o n anticodon interaction prior to the hydrolysis of GTP does not provide sufficient discrimination to ensure that translation takes place with sufficient accuracy at the high working speed required [1,2], and the term 'proofreading' is used to cover the various mechanisms for multiple-checking that have been postulated both for the translation of RNA into protein and for other biological copying processes. One way in which proofreading can occur in the translation process is the allowance of the possibility that aa-tRNA may dissociate from the ribosome, after GTP hydrolysis, with a dissociation rate that is higher for aa-tRNAs that are not cognate to the codon, that is, whose energy of interaction with the A site is low, since the discrimination lies in the off-rate [3, 4]. Roughly speaking, we can say that proofreading of A site-bound aa-tRNA occurs if the following inequality holds: rate (dissociation if non-cognate)>rate (next step towards peptidyl transferase)> rate (dissociation if cognate). This concept has been refined and investigated thoroughly in the past decade, and the theoretical and experimental contributions that have been made in this field are reviewed in depth l i t [,,31, t ~ - ' ~ | d J L l ~
[2].
|11 L i l t ~ l l i ~ p t U l
oy
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Antibiotics that act on the ribosome have frequently been used for the investigation of individual steps of protein biosynthesis. Numerous antibiotics are known that interfere with the binding of the ternary complex ( a a - t R N A - E F - T u - G T P ) in the elongation cycle, including many that induce misreading [6-8]. Work by Davies, Gorini and others [6, 9, 10] suggested that these antibiotics induce misreading by causing structural changes at the A site. Spirin [11] and Suzuki et al. [12] suggested that they might function by stabilising the 30S-50S interaction. However, this hypothesis was refuted by the discovery [13] of a streptomycin analo~e that does stabilise this interaction but does not induce misreading. Investigations by Ninio [4] and Gavrilova et al. [14] lent weight to the interpretation that the misreading-inducing aminoglycosides raise the speed and thus the error of protein biosynthesis. Kurland [15] emphasized the possibility that these antibiotics might affect not the site of codon-anticodon contact, but some other element of tRNA-ribosome interaction at the A site.
In earlier work [16-18], we investigated the effects of various kinds of perturbation on the prokaryotic (E. coil) ribosomal complex (70SAUGUUU-tRNAMet-Phe-tRNAPhe). The complex was prepared by the enzymic route (i.e., the A site was occupied by Phe-tRNA Phe with the help of EF-Tu and GTP), and the perturbations applied were" (1) shortening or lengthening of the UUU codon; (2)interruption of the hexanucleotide messenger, using, e.g., AUG and UUU separately; (3) replacement of one of the bases in the UUU triplet by a different base; and (4) addition of an antibiotic, viomycin (which strengthens binding of aa-tRNA in the A site) or sparsomycin (which weakens it). The behaviour of the complex in all these situations was explicable by the hypothesis that the fate of the bound aa-tRNA, after hydrolysis of GTP and before peptidyl transfer, is determined principally by the energy of binding of the aa-tRNA to the A site. This energy could be decreased by a shortened or mutated codon, an interruption of the messenger (promoting dissociation) or a destabilising antibiotic. The behaviour of the 70S complex with occupied A site was observed, using three experimental criteria" (1)the stability of the A site binding, (2) the turnover of GTP, and (3) the amount of dipeptide formed when the P site contained fMettRNA Met instead of uncharged tRNA Met. It was found that a small decrease in binding energy did not greatly reduce the ability of (EFT u - ~ l r - r n e - t R N A Phe) to bind to the A site and undergo GTP hydrolysis, but it resulted in the PhetRNA abe dissociating again and vacating the A site. Thus, while the normal binding energy resulted in stoichiometric binding of Phe-tRNA ehe to the ribosome and a low GTP turnover, binding weakened, e.g., by a single mismatching base in the codon gave a low binding level of Phe-tRNA ehe and a greatly increased GTP turnover. Dipeptide formation was rapid and quantitative under conditions of normal binding, and with weakened binding the amount of dipeptide was small, and rose slowly and linearly, as would be expected from the recycling of Phe-tRNA abe. Further weakening of the interaction (e.g., two codon bases missing) resulted in zero binding of Phe-tRNA Phe and a low or zero GTP turnover. The effect of one mismatching base in the codon could be simulated by the antibiotic sparsomycin, and it could be reversed by viomycin. Thus the response of A site-bound aa-tRNA was, empirically, largely reducible to a single parameter" the energy of the interaction between the aa-tRNA and the A site before and after GTP hydrolysis.
Antibiotics and misreading at the ribosomal A site Detailed effects, such as those observed with the non-cognate codon CUU, which gives a weak but non-productive specific stabilisation of PhetRNA Phe in the A site [18], do not affect qualitatively the general conclusion. The effects of perturbation of aa-tRNA in the A site could thus, in the first instance, be reduced to increments and decrements of its binding energy. If the binding energy is a variable dependent upon the sum of many, approximately independent interactions, then it should be possible to extend the range of stabilising and destabilising factors and to see to what extent they can be made to reinforce one another or to cancel out. The purpose of this investigation was therefore to find out how other antibiotics influence this test system, and to what extent their behaviour can also be rationalised in this simple way.
Materials and methods The isolation and purification of tight-couple 70S ribosomes from E. coli followed standard methods [19]. The preparation and characterisation of oligonucleotides, the charging of tRNA and the assay conditions for aa-tRNA binding, phosphate turnover and dipeptide formation have been described elsewhere [16-18, 20]. In summary, the assays were conducted as follows, all at 37°C. 25 pmol of 70S ribosomes, 50 pmol of deacylated tRNAMet and 2 nmol of messenger oligoribonucleotide were pre-incubated for 2 min in 75 pl of the standard h n f f e r , n ~ d in pre~ous ~tl,d;,~ ~fis/Tris hydrochloride 25 mM, pH 7.5, ammonium chloride 50 mM, 2-mercaptoethanol 2.5 mM and magnesium acetate 11 mM or, in certain cases where stated, 5 raM). If required, antibiotic was added in 5/zl of dimethyl sulphoxide and the pre-incubation was repeated. To start the reaction, 25 pl of a mixture of EF-Tu (120 pmol), GTP (1 nmol) and [3H]aa-tRNA, pre-incubated in the same buffer, was added. For the assay of aa-tRNA binding, the amount of radioactivity bound to the ribosomes was determined by a Millipore filter test. For dipeptide synthesis, deacylated t R N A Met in the above test was replaced by fMet-tRNAfMet and the amount of radioactive dipeptide formed was measured by extraction into ethyl acetate. For the assay of GTP hydrolysis, [7'-32p]GTP (0.5 Ci/mmol), phosphoenol pyruvate (2 mM) and pyruvate kinase (0.1 mg/ml) were also included, the reaction was stopped with perchloric acid and the inorganic phosphate liberated was determined by extraction with molybdate into isopropyl acetate. Antibiotics were purchased from Sigma (Munich); several were kindly donated by Dr. E Cundiiffe (Leicester University). The concentrations of antibiotics in the assays were reasonably high (greater than the dissociation constants, where these are known), so the ribosomes may be considered as being saturated with antibiotic, or nearly so.
805
Results Antibiotics that induce misreading The occupation of the A site by Phe-tRNA Phe was monitored as a function of the length of the A site codon in the presence of various antibiotics. Fig. I shows typical results. On the left, the complete codon UUU gives a plateau of Phe-tRNA Phe binding and phosphate consumption (A). Weakening of the A site affinity for Phe-tRNA Phe, by use of the truncated codon UU, gives a low plateau and a high phosphate consumption, which are due to dissociation and continual recycling of PhetRNA Phe (B). Further weakening (truncated codon U) means that the residence time of the ternary complex is reduced so much that GTP hydrolysis hardly takes place (C). On the right, the stabilising effect of neomycin is seen. While the antibiotic has only a slight effect on the already stable complex with the triplet codon, the weakness of the UU codon is compensated for (E). Even the extremely weakly-binding codon U is strengthened a little, so that more GTP is hydrolysed, even though all the Phe-tRNA Phe dissociates again (F). Similar results were o b t a i n e d with the misreading-inducing aminoglycoside antibiotics streptomycin, kanamycin and gentamicin and were also indicated in an earlier study with viomycin [17]. However, the aminoglycoside antibiotic spectinomycin, which does not induce misreading, had no effect in these experiments, the results of which are summarised in Table I, columns 1-3. All the misreading-inducing antiobiotics tested provide a stabilisation of aa-tRNA in the A site that is roughly equivalent to the destabilisation caused by a missing codon base. Two missing codon bases can only be compensated for to a small extent. Although the stability of A site binding is influenced, its geometry cannot be grossly distorted, as is shown by the almost invariant yield of dipeptide (80-85°7o). The loss of one base from the codon is thus compensated for by the presence of a misreadinginducing antibiotic. The fact that the loss of two bases is not compensated, so as to appear like the loss of only one, suggests the possibility that the stabilising effect of these antibiotics may be effective only after GTP hydrolysis. However, this is not the case, as shown by repeating the experiment replacing GTP by its non-hydrolysable analogue GDPNP. It is already known that when cognate ternary complex with GDPNP is offered to the A site, initial binding takes place, but the EF-Tu is not discharged from the 70S complex, and the
806
H. Hornig et al.
aminoacylated 3' end of the tRNA does not take part in the peptidyl transferase reaction [21]. The effect of replacing GTP with GDPNP is shown in Table I (4th column): the stabilisation of A site-bound Phe-tRNA Phe by misreadinginducing antibiotics also occurs when the Phe-
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tRNA Phe is bound to the A site in its pre-GTPhydrolysis conformation, along with EF-Tu. In the presence of the non-hydrolysable GTP analogue, none of the Phe-tRNA Phe is able to adopt its postGTP-hydrolysis conformation, as shown by the absence of dipeptide formation, found for all an-
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8
Time(rain)
Fig. 1. P site complexes were prepared by incubation of 70S ribosomes and tRNA~ et, with the oligoribonucleotide AUG(U)n fis messenger. At time zero, ternary complex (EF-Tu-GTP-Phe-tRNAPhe) was added and the course of Phe-tRNA Phe binding and GTP hydrolysis was monitored. A : messenger = A U G U U U ; B: A U G U U ; C : A U G U . D - F , same as A - C , but 100/zM neomycin was added to the pre-incubation mixture (see Materials and Methods).
Antibiotics and misreading at the ribosomal A site tibiotics (the 1°70 observed is not significant). Again, neomycin is able to compensate for the loss of one base in the codon (Table I, 5th column). In all these experiments, spectinomycin has no stabilising or destabilising effect, which confirms that the stabilisation by these antibiotics is indeed correlated with their ability to induce misreading. The same effect is observed when, instead of a s h o r t e n e d c o d o n , a n o n - c o g n a t e c o d o n is employed. A single base substitution has an effect roughly equal to that of a single deletion, and, as Table II shows, this decrease in binding affinity can be made good by the addition of neomycin. Again,
807
there is no impairment of the ability of Phet R N A Phe, once bound, to form dipeptide. In Table II, the stoichiometric ratio of G T P hydrolysed to P h e - t R N A Phe bound should be noted. For stable binding, fewer than 2 G T P molecules are hydrolysed per Phe-tRNAl'he: for unstable binding, this ratio exceeds 30 during the 3 min incubation and continues to rise thereafter. Another way of weakening the A site binding of a a - t R N A is to interrupt the hexanucleotide messenger, by using for example (AUG + UUU) or ( A U G U + UU) [16]. Although this weakening has no counterpart in vivo, its effects are of con-
Table I. Yields of bound Phe-tRNA l'he and dipeptide for various antibiotics, for normal and shortened A site codons and for GTP/GDPNP.
Antibiotic
AUGUUU, GTP
AUGUU, GTP
AUGU, GTP
AUGUUU, GDPNP
AUGUU, GDPNP
None Neomycin Gentamicin Kanamycin Streptomycin Spectinomycin
68 (85) 74 (85) 74 72 72 68
8 72 70 68 68 8
4 (< 1) 7 (< 1) 7 6 6 4
20 76 74 74 70 20
4 12 4
(> 100)a (84) (81) (83) (81)
(1) (1) (1) (1) (1) (1)
70S ribosomes (25 pmol) were programmed at the P site by incubation with messenger and tRNA~et (for the A site binding assay) or fMet-tRNA~et (for the dipeptide assay). After addition of 100 tzM antibiotic, if appropriate, the required ternary complex (EFTu and Phe-tRNATM pre-incubated together with GTP or GDPNP) was added. After reaction to completion (3 min; in some eases 10 min was allowed) the binding or dipeptide measurement was made [16]. The yield of bound Phe-tRNApheis stated as a percentage of the number of ribosomes (maximum obtainable = 75070). The yield of dipeptide, where measured, is stated in parentheses as a percentage of the amount of bound Phe-tRNAPh% a The percentage dipeptide yield has no meaning this case, since, in accordance with the steady-state binding pattern, weak A site occupation results in a slow, linear accumulation of dipeptide, to give a 'yield' which increases steadily and, at length, exceeds 100070 w.r.t, bound Phe-tRNAPhe.
Table II. Yields of bound Phe-tRNA Phe and dipeptide for various antibodies and altered A site codons.
Oligonucleotide
AUGUUU AUGCUU AUGUU AUGUCU AUGUUG AUGGUU AUG + UUU AUG + UUU AUG+UUU AUG + UUU
No antibiotic
+ Antibiotic
Antibiotic
Yield
GTP hydrolysed
Yield
GTP hydrolysed
68 (85) 22 8 7 6 4 1.6 1.6 1.6 1.6
128 108 260 252 256 108 <2 <2 <2 <2
74 (85) 66 (78) 65 (84) 56 (80) 62 (83) 48 (76) 40 (78) 0.6 (= 70) 0.6 (=80) 0.4 (= 75)
120 115 120 153 144 110 120 160 160 <5
neomycin neomycin neomycin neomycin neomycin neomycin neomycin gentamicin kanamycin streptomycin
The neomycin concentration was 100 btM. Yield of bound Phe-tRNAPheand turnover of GTP are given as aT0of ribosomes present; dipeptide yield, where measured, is given in parentheses as 070of bound Phe-tRNAphe.
H. Hornig et al.
S08
tion, so results are presented both for 11 mM and 5 mM Mg 2+ (Table III, 1st and 2nd columns). Four of them showed no inhibitory effect and were not investigated further. The remaining nine were divided into categories, according to their ability to suppress as well the binding of the tertiary complex ( E F - T u GDPNP-Phe-tRNAPhe), which contains the nonhydrolysable analogue of GTP. Only two of them, chlortetracycline and thiostrepton, suppressed its binding. The remaining seven are described in the literature as peptidyl transferase inhibitors, and our measurements of dipeptide formation (Table III) agree with this. Yet they clearly have a further influence, as shown by an earlier experiment [17] with sparsomycin, whose results we recapitulate briefly. If the time course of Phe-tRNA Phe binding and GTP hydrolysis are measured as in Fig. 1, then the following pattern emerges. (1) For the messenger AUGUUU, sparsomycin lowers the plateau level of bound Phe-tRNA Phe from 0.65 mol/ribosome (Fig. 1A) to about 0.17 mol/ribosome (Fig. 2D). However, it remains a clear plateau. In contrast, the phosphate turnover is more than doubled and
siderable interest. Like the other kinds of destabilisation (above), this weakening was compensated for by neomycin, an effect which we have already observed for viomycin [16]. Interestingly, gentamicin and kanamycin have a weaker influence and streptomycin does not appear to work at all (Table II). There is already a hint (Table II) that neomycin exerts a stronger stabilising effect, and streptomycin a weaker one, than do the others. Apparently the interruption of the messenger has a greater destabilising influence and thus calls for a greater stabilisation, so that it discriminates more strongly between the degrees of stabilisation offered by the different antibiotics. The stronger discrimination may also be due to a different site of action, e.g., with the site of interaction of neomycin being closest to the anticodon.
Antibiotics that inhibit peptidyl transfer Thirteen antibiotics frequently described as being A site active were tested for their ability to inhibit the binding of Phe-tRNA abe to the cognately programmed A site. It was observed that some had a greater effect at lower magnesium ion concentra-
Table HI. Inhibition of binding of Phe-tRNA phe in the A site programmed by A U G U U U , in the presence of GTP or G D P N P . A ~.=I.=~:~ J"kJ[l LI UIU
V:~l_l Lllb,
• ,wu
1~ . . . .
..I
u u u . u
n k -
r
.
,.n~!
~
-
t
m
•
~
PhP
....
___'~L
w~m G T P
'1[/'o _!21
_
~lffb I ~ T 4, ] [ ~ h o ....
11 mM Mg 2+
5 mM Mg 2+
None
80
(82)
38
(82)
63
Chloramphenicol Streptogramin Sparsomycin Niddamycin Carbomycin Clindamycin Lincomycin
35 20 28 31 30 61 62
( = 1) ( --- 1) ( = 1) ( = 1) ( = 1) ( = 1) ( = 1)
15 4 11 12 13 14 15
( = l) ( = 1) ( = 1) (=1) (=1) (--- l)
62 64 63 65 62 64 61
Chlortetracycline Thiostrepton
27 72
3 9
Erythromycin Thylosin Spiramycin Bottromycin
77 80 77 79
38 39 39 37
(----l)
'I[~'L
I l t ~ l U bound r ' n e - t ~ ' ~ with G D P N P (5 mM Mg 2+)
10 11
Reactions were carried out at two Mg2+ concentrations (see text). Concentrations of antibiotics were: chlortetracycline: 150/zM; thiostrepton: 21/zM ; streptogramin and sparsomycin: 55/zM; others: 100/zM. Incubation time was 3 min. Yield of bound PhetRNA phe is given as o70 of ribosomes present: dipeptide yield, where measured, is stated in parentheses as % of bound Phe-tRNA phc. The messenger was AUGUUU for the experiments with tertiary complex including GTP and poly(U) with tRNA phe in the P site for those including GDPNP.
Antibiotics and misreading at the ribosomal A site
oE°0.4S ~r o 0.3
1.5 oE
B
A
1.0
L
0.2
"rI/} D u
0.5
_~ 0.1 o
o
E "0
o ..13
E tJ
'
I
'
D
I
'
C
o e~
I
'
I
0
0.8
O f,,.. "13 t-
< 0.3
zn,,. .,-, 0.2 I .c a. 0.1
"tO Q,I U}
1.0
13_ 1--
0.5-
~"~~
1
13_
"I"
=
0
I
0
-
4
;
-,
8
,
0
,
4
,
,
8
Time (rain) Fig. 2. Time course of Phe-tRNA Phe binding and GTP hydrolysis in the presence of the messenger AUGUUU (cf. Fig. IA). A: no antibiotic; B: plus 150/zM chlortetracycline; C: plus 21 btM thiostrepton; D: plus 55 /zM sparsomycin [17]. Measurements A - C were made at a lower Mg 2+ concentration (see text), which depresses all binding levels.
does not reach a plateau, but continues to rise. (2) For the messenger AUGUU, which gives a very low steady-state binding level of Phe-tRNA Phe and a high, rising GTP turnover (Fig. IB), the addition of sparsomycin has no effect (not illustrated). Both of these observations indicate that the inhibiting effect of sparsomycin at the peptidyl transferase site is accompanied by a physical (presumably steric) blockage that also interferes with, and destabilises, the binding of the aminoacylated CCA end of the A site-bound tRNA. This has less destabilising effect on the A site aa-tRNA molecules than, for example, a noncognate codon (compare Fig. 1B and 2D), but it nevertheless leads to an elevated rate of dissociation of aa-tRNA from the A site and thus both to a lower stationary-state binding plateau and to a higher, continuing consumption of GTP.
Antibiotics that block binding o f the ternary complex Of all the antibiotics tested, only two, tetracycline and thiostrepton, were found to depress the bind-
809
ing level of cognate ternary complex containing the non-hydrolysable GTP analogue (Table lII). Both of these are known to be A site inhibitors [22], but there have been contradictory ideas about their mechanism of action [23-25] and, in the case of tetracycline, its binding site (reviewed in [26]). We therefore subjected them to closer investigation. They both inhibited Phe-tRNA ehe binding more efficiently at a lower magnesium concentration, so most measurements are made both at standard (11 raM) and low (5 raM) magnesium concentrations. Although these inhibitors weaken the interaction between the A site and Phe-tRNA vhe, it is clear that they do this in a way different from that of mismatching bases and other inhibitors such as sparsomycin (Table IV and Fig. 2). This is because a small destabilisation leads not to a higher, but to a lower phosphate consumption, which does not proceed indefinitely but rapidly reaches a plateau, at a level depressed in proportion to the depression of the Phe-tRNA Phe binding level. Another difference between these two antibiotics and sparsomycin is their effect on the behaviour of A sites programmed with an incomplete triplet (messenger: AUGUU, cf. Fig. 1B). As mentioned, sparsomycin has virtually no effect on this system. However, if thiostrepton is included in the experiment described in Fig. IB, then the phosphate turnover drops to 25070, and to < 5°70 in the presence of chlortetracycline. This is clearly consistent with the observation (Table III) that these antibiotics inhibit the approach of ternary complex, since this naturally results in a drop in the amount of GTP hydrolysed. This drop is not due to inhibition of the actual GTP hydrolysis, since this GTPase activity is not blocked by thiostrepton [27].
Effects o f simultaneously-bound antibiotics If the concept of incremental stabilisation and destabilisation of Phe-tRNA Phe in the A site is correct, then it follows as a corollary that different antibiotics, acting simultaneously at different points on the A site complex, should tend to reinforce each other or to cancel each other out. With the large numbers of antibiotics tested, this suggests many possible combinations with which to experiment. However, in order to test the concept, we restricted ,this investigation to combinations of one representative from each group. These were: neomycin, for the antibiotics that induce misreading and stabilise aa-tRNA in the A site; streptogramin, for the antibiotics that inhibit the peptidyl transferase centre and destabilise the 3' terminus; and chlortetracycline, for the antibiotics that interfere with
H. Hornig et al.
810
the binding of the ternary complex to the A site prior to GTP hydrolysis. The effects of combining neomycin and streptogramin are shown in Table V (upper half). As expected, streptogramin suppresses and neomycin stimulates the binding of Phe-tRNA Phe to the A site. When both are present, either with AUGUUU or with the truncated AUGUU as messenger, the combined binding effect is largely the same as that seen when neither is present. However, the presence of streptogramin also abolishes the peptidyl transferase reaction.
T a b l e I V . Y i e l d o f b o u n d P h e - t R N A Phe,
active against ternary complex Yield
Antibiotic
stimulation of GTP hydrolysis and dipeptide yield in the presence of antibiotics
binding.
of bound
11 m M
None Thiostrepton Chlortetracycline
The combination of chlortetracycline and neomycin gave similar results (Table V, lower half). This is of greater significance, because it shows that the stabilisation by neomycin operates on aa-tRNA in the A site not only after hydrolysis of CTP, but also before. Further, it was shown that spectinomycin had no effect in conjunction with chlortetracycline, just as it had none alone in these assays (Table I). This strengthens our earlier deduction that spectinomycin has a mode of action different from that of the other aminoglycosides.
Phe-tRNA
M g 2+
5 mM
80 (82)
Mol GTP
Phe M g 2+
11 m M
38 9 3
72 (80) 28 (79)
•
•
lt~ll~V~.,L~
Vl
~IIIIUILCI.I£1wUUO
lJll,,Ol,,,aal.,~,.
Va
M g 2+
5 mM
128 120 43
~aaL~.~.,JaaaoLx~.~aaj-r.,~,v.xaz~
1Jr.Laao
M g 2+
68 13 6
Bound P h e - t R N A Phe a n d G T P hydrolysis is expressed in molecules per 100 ribosomes age of bound P h e - t R N A Phe.
la~UlIK;
hydrolysed
vl
and dipeptide,
in parentheses as the percent-
r.a~xal.xvJaVl.a-~o.
Oligonucleotide
Streptogramin
Neomycin
Chlortetracycline
Yield bound
AUGUUU AUGUUU AUGUUU AUGUUU
+ +
+ +
-
80 8 84 68
(80) (1) (82) (1)
AUGUU AUGUU AUGUU AUGUU
+ +
+ +
-
8 2 80 19
(84)
AUGUUU AUGUUU AUGUUU AUGUUU
-
+ +
+ +
80 20 84 76
(83) (79) (82) (82)
AUGUU AUGUU AUGUU AUGUU
-
+ +
+ +
8 4 80 10
Phe-tRNA
(84)
Incubation was under standard conditions, 11 m M M g 2+. Antibiotic concentrations were as in T a b l e l l I . The yield of bound percentage of ribosomes present, the yield of dipeptide in parentheses as a percentage of bound spectinomycin to the binding assay with or without chlortetracycline had no effect.
t R N A Phe is expressed as a t R N A TM. The addition of
Phe
PhePhe-
Antibiotics and misreading at the ribosomal A site
Discussion Fourteen of the nineteen antibiotics studied can be classified into three groups, according to their effects on aa-tRNA bound at the ribosomal A site. I. Neomycin, viomycin, gentamicin, kanamycin and streptomycin stabilise aa-tRNA in the A site both before and after GTP hydrolysis. By doing this, they cause errors, in particular in proofreading (dissociation of non-cognate aa-tRNA after GTP hydrolysis). They appear to have a stabilising effect which diminishes in the above order, as can be seen in Table I. (The placing of viomycin is based on its ability [16] to stabilise aa-tRNA effectively in the presence of an interrupted messenger, which otherwise only neomycin was able to do; see Table II.) It is worthy of note that both neomycin and viomycin, apparently the strongest stabilisers of aa-tRNA in the A site, are regarded not only as inducers of misreading, but also as inhibitors of translocation ([28], reviewed in [29]). If they cause aa-tRNA to bind tightly to the A site, then inhibition of translocation, to some degree, is a necessary consequence. No member of this group has any influence on the peptidyl transferase centre, whose efficiency is unimpaired by their presence. II. Chlortetracycline and thiostrepton interfere with the binding of ternary complex. It is consistent with their destabilising, rather than stabilising, ,,, ,~,~-,.,,,,.~ . . t , , ~ A ~tc that neither oz these antibiotics induces misreading. For both antibiotics, GTP hydrolysis is reduced in proportion to the reduced aa-tRNA binding (Fig. 2), while the peptidyl transferase reaction is unaffected (Table IV). The latter finding has also been reported for tetracycline by others [30]. The fact that chlortetracycline and thiostrepton have the same effect is hard to interpret structurally, because the presumed sites for tetracycline and thiostrepton binding are very far apart on the ribosome. The former antibiotic binds to protein $7 (as shown by photolytic cross-linking[31]), close to a recent placing of the A site [32], but the latter binds to a region of the 23S RNA that is thought to be some 100 ~. away from this [33]. Using the fluorescence anisotropy changes associated with the immobilisation of tetracycline on binding to ribosomes [26], we have shown that the affinity of the A site for Phe-tRNA Phe is reduced by some 80070, if tetracycline is already bound to its strong binding site on the 30S subunit [34]. This applies for both enzymic and non-enzymic occupation of the A site. It thus appears that the
811
suppression of the binding plateau (without destabilisation of A site-bound Phe-tRNA Phe) in Fig. 2 is due simply to effective non-availability of A sites of ribosomes to which a tetracycline molecule is already bound. The division of the population of ribosomes by tetracycline into 'available' and 'non-available' is consistent with our observation [26] that the tetracycline-ribosome binding takes a long time to equilibrate. This argument cannot apply to thiostrepton, which has a very high constant of binding to ribosomes [35] and yet puts only = 75°7o of them out action (Fig. 2; Table IV), so that (especially in view of the structural consideration above) there may be some differences in the mechanism that our data do not reveal directly. III. Streptomycin, sparsomycin, niddamycin, carbomycin, chloramphenicol, clindamycin and lincomycin act as antibioticsby abolishing totallythe activityof the peptidyl transfcrasecentre. But they also destabilisethe aa-tRNA bound in the A site, which makes them of interestas tools for studying aa-tRNA-ribosome binding. The strongest destabifiserswere found to be streptogramin and sparsomycin, which are also the only inhibitorsof P h e - t R N A binding in the Phe-tRNA/poly(U) binding test [22,36, 37]. Of the antibioticstested,only crythromycin, bottromycin, thylosin, spiramycin and spectinomycin had no effect on A site-bound aa-tRNA. Of these, erythromycin does not inhibitpeptidyl transferper se [35], which agrees with our data if it acts without affecting the A site. Bottromycin is claimed to inhibit the binding of aa-tRNA to the A site [22], an assertion which our data throw doubt on, since no effect of bottromycin on aa-tRNA binding was observed (Table III). However, the purpose of these experiments was less to investigate the mechanisms of action of the antibiotics than to investigate the connection between classes of antibiotic and the behaviour of aa-tRNA in the A site, setting this in relation to other stabi!t~singand destabilising factors. It is also clear that t,~lelethal quality of the antibiotics is not necessarily connected with the stability of bound aa-tRNA: for example, the destabilisation by sparsomycin and others is clearly incidental to peptidyl transferase inhibition, while gentamicin has a complex spectrum of effects [22]. None the less, it is interesting that none of the nineteen antibiotics tested destabilised aa-tRNA after GTP hydrolysis only. This may suggest that the structural difference in A site tRNA before and after GTP hydrolysis is not very great. The hypothesis formulated earlier [18], that the behaviour of aa-tRNA in the A site is determined
812
H. Hornig et al.
by the requirementof 'correct' binding energy, and that various structural factors contribute energetic increments or decrements, appears to be supported. Decrements are provided by (l) incorrect binding of the aminoacylated 3' end of the tRNA due to the presence of a peptidyl transferase inhibitor; (2) a missing codon base; (3) a non-cognate codon base, where the size of the decrement depends on the 'badness' of the mismatch; (4) destabilisation of the messenger, for example by interruption of the sugar-phosphate chain, which promotes dissociation; and (5) A site-destabilising antibiotics. Increments are supplied by certain stabilising antibiotics. These conclusions are in good agreement with the results of other groups, including the observation of weakened binding of tRNA non-cognate to the A site in the presence of the messenger poly(U) [28] with accompanying stimulated GTP hydrolysis [38-41] and stabilisation by neomycin or streptomycin [42-44]. In each case, the result of what we would term a binding energy decrement (one incorrect base pair) or increment (misreadinginducing antibiotic) corresponds with that found in the present work. The energetic increments and decrements can be balanced off against one another so that they cancel out. It is particularly interesting that this should be possible for the simultaneous addition of different antibiotics, since it lends support to the picture of separate and largely independent points of action for many antibiotics. This is supported tentatively by the observation of different points of binding of the misreading-inducing antibiotics, e.g., streptomycin and neomycin [29, 45]. The pursuit of combination experiments, such as presented in Table V, may lead to more information about the mechanisms of action of various antibiotics. The fact that antibiotics with the stabilising propetty are precisely those that induce misreading provides strong conf'mnation of the hypothesis that the ability of aa-tRNA to dissociate from the A site after GTP hydrolysis, if its binding energy is lower than normal by a decrement corresponding to a single codon-anticodon mismatch, is a part of the mechanism ensuring ribosomal accuracy. The effect of losing an AU base pair in the binding of PhetRNA phe is about the same as that of losing a GC base pair in the binding of Pro-tRNA Pr° [17], confirming that not just the codon but the whole tRNA molecule has evolved so as to bring the total energy of binding of each individual tRNA species to precisely the right value [46]. There is increasing evidence that streptomycinresistant mutant ribosomes have, in vitro, a greater
missense suppression, a higher accuracy and a higher GTP turnover per peptide bond made than do wild type ribosomes (reviewed in [2]). In the context of the present work, this would suggest that the mutant has an A site with lowered affinity for all aa-tRNAs after GTP hydrolysis (perhaps also before GTP hydrolysis, but this would not necessarily affect the accuracy). It would be interesting to assay such ribosomes in vitro in the manner described here, so as to investigate the magnitude of the decrement and its (anticipated) generality for all tRNA species.
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A n t i b i o t i c s a n d misreading at the ribosomal A site
19 NoU M., Hapke B., Schreier M.H. & Noll H. (1973) J. Mol. Biol. 75, 281-294 20 Hornig H. (1984) Dissertation: Mechanismus der aa-tRNA Bindung, Technische Universit~it, Berlin 21 Kaziro Y. (1978) Biochim. Biophys. Acta 505, 95-127 22 Cundliffe E. (1980) in: Ribosomes: Structure, Function and Genetics (Chambliss G., Craven G.R., Davies J., Davis K., Kahan L. & Nomura M., eds.), University Park Press, Baltimore, pp. 555-581 23 Gordon J. (1969) J. Biol. Chem. 244, 5680-5686 24 Kinoshita T., Liou Y.-F. & Tanaka N. (1971) Biochem. Biophys. Res. Commun. 29, 356-361 25 Modolell F., Cabrer B., Parmeggiani A. & Vasquez D. (1971)Proc. Natl. Acad. Sci. USA 68, 1796-1800 26 Epe B. & Woolley P. (1984) EMBO J. 3, 121-126 27 Ballesta J.P.G. & Vasquez D. (1972) Proc. Natl. Acad. Sc~. USA 69, 3058-3062 28 Cabafias M.J. & Modolell J. (1980) Biochemistry 19, 5411-5416 29 Gale E.F., Cundliffe E., Reynolds P.E.; Richmond M.H. & Waxing M.H. (1981) in: The Molecular Basis o f Antibiotic Action, Wiley, London, pp. 402-547 30 Semenkov Y.P., Makarov E.M., Mahnko V.I. & Kirrilov S.V. (1982) FEBS Lett. 144, 125-129 31 Goldman R.A., Hasan T., Hall C.C., Strycharz W.A. & Cooperman B.S. (1983) Biochemistry 22, 359-368 32 Ofengand J., Ciesiolka J., Denman R. & Nurse K. (1986) in: Structure, Function and Genetics o f Ribosomes (Hardesty B. & Kramer G., eds.), Springer-Verlag, New York, pp. 473-494
813
33 St6ffler G. & St6ffler-Meilicke M. (1986) in: Structure, Function and Genetics o f Ribosomes (Hardesty B. & Kramer G., eds.), Springer-Verlag, New York, pp. 28-46 34 Epe B., Woolley P. & Hornig H. (1987) FEBS Lett. 213, 443-447 35 Cundliffe E., (1986) in: Structure, Function and Genetics o f Ribosomes (Hardesty B. & Kramer G., eds.), Springer-Verlag, New York, pp. 586-604 36 Ravel J.M., Shorey R.L. & Shive W. (1970) Biochemistry 9, 5028-5033 37 Chinali G., Sprinzl M., Parmeggiani A. & Cramer F. (1974) Biochemistry 13, 3001-3010 38 Thompson R.C., Dix D.B., Gerson R.B. & Karim A.M. (1981) J. Biol. Chem. 256, 81-86 39 Thompson R.C. & Stone P. 0977) Proc. Natl. Acad. Sci. USA 74, 198-202 40 Donner D., ViUems R. & Kurland C.G. (1978)Proc. Natl. Acad. Sci. USA 75, 3192-3195 41 Ruusala T., Ehrenberg M. & Kurland C.G. (1982) EMBO J., 1 741-745 42 Yates J.L. (1979) J. Biol. Chem. 254, 11550-11554 43 Thompson R.C., Dix D.B., Gerson R.B. & Karim A.M. (1981) J. Biol. Chem. 256, 6676-6681 44 Campuzano S., Cabafias M.J. & Modolell J. (1979) Eur. J. Biochem. t00, 133-139 45 Gris6-Miron L., Noreau J., Melancon P. & BrakierGingras L. (1981) Biochim. Biophys. Acta 656, 103-110 46 Ninio J. & Claverie P. (1971) J. Mol. Biol. 56, 63-82