Eur. J. Biochem. 98, 261 -265 (1979)

Binding of Thiostrepton to a Complex of 23-S rRNA with Ribosomal Protein L11 Jill THOMPSON, Eric CUNDLIFFE, and Michael STARK

Department of Biochemistry, University of Leicester

(Received March 8, 1979)

Thiostrepton binds with high affinity and with a 1 : 1 stoichiometry to a complex formed be- tween Escherichia coli 23-S ribosomal RNA and ribosomal protein L11 of E. coli or the homologous protein BM-L11 of Bacillus megaterium.

In the presence of T1 ribonuclease, protein BM-L11 and thiostrepton protect from degradation a fragment of E. coli 23-S RNA estimated to be about 50 nucleotides in length.

Thiostrepton inhibits bacterial protein synthesis by binding with high affinity to the 50-S ribosomal subunit with 1 : 1 stoichiometry. As a result the drug inhibits, in various assay systems in vitro, component reactions of protein synthesis involving the hydrolysis of GTP in association with the protein factors EF-G, EF-Tu and IF-2 (for a review, see [l]). High-affinity binding of thiostrepton to Escherichia coli ribosomes requires the presence of ribosomal protein L11 and this single protein also restores drug-binding to 4 M LiCl core particles derived from 50-S ribosomal sub- units [2]. We have recently observed a similarity in ribosomes of Bacillus megaterium where a protein ‘BM-L1 l’, serologically homologous to protein L11 of E. coli, is required to create or complete a high- affinity binding site for thiostrepton [3]. Also we have examined the ribosomes of a number of thiostrepton- resistant mutants of B. megaterium (e.g. mutant MJI) and have demonstrated by immunological techniques that they lack protein BM-L1 1. Such ribosomes do not possess a high-affinity site for thiostrepton but, evidently, still retain low affinity for the drug since they remain sensitive to its action both in vitro and it? vivo, albeit at elevated drug concentrations.

Here we present the results of recent attempts to determine more precisely the ribosomal components involved in the high-affinity binding site for thio- strepton. Thiostrepton is particularly suitable for use in the these studies since there are obvious advantages in being able to observe directly the binding to ribo- somal components of parent antibiotic molecules as opposed to synthetic affinity analogues. Problems inherent in the use of the latter have been critically discussed elsewhere [4].

Ei?;jmes. Tl ribonuclease or T1 RNase (EC 3.1.4.8); pan- creatic ribonuclease or RNase I (EC 3.1.4.22).

MATERIALS AND METHODS

Preparation of Ribosomal Proteins

Pure protein L11 of E. coli was kindly provided by Dr Georg Stoffler. This protein had been prepared by ion-exchange chromatography on carboxymethyl- cellulose in phosphate buffer containing 6 M urea at pH 6.5 followed by gel filtration on Sephadex G-100 in similar buffer.

Protein BM-L1 1 of B. megaterium was resolved from others present in the 1 M LiCl split protein fraction by ion-exchange chromatography on carboxy- methyl-cellulose as described elsewhere [3] with the modification that urea was omitted from the buffers employed.

Ribosomal core particles and split proteins were prepared as described fully elsewhere [3].

Preparation of Ribosomal R N A

Total ribosomal RNA was extracted with phenol from 70-S ribosomes of B. megaterium by a standard procedure [5] and was routinely dissolved in buffer A and stored at - 70’C. Buffer A contained 10 mM Tris-HCI pH 7.6, 10 mM MgClz, 50 mM NH4CI and 3 mM 2-mercaptoethanol. Components of total ribo- somal RNA were obtained by minor modifications of the technique of Harrison et al. [6], which involves extraction of ribosomes with phenol and sodium dodecylsulphate followed by sucrose density centrif- ugation. RNA fractions from each ultraviolet-absorb- ing peak were pooled and re-extracted with phenol as described for total rRNA.

[35S]Thiostrepton was prepared as described pre- viously [7] and was used in these experiments at a specific radioactivity within the range 350 - 250 counts min-’ pmol-’.

262 Thiostrepton Binding Site

Assays for the Binding of(35S]Thiostrepton to RNA-Protein Complexes

These were usually carried out in 100 p1 total volume in standard incubation buffer containing 10 mM Tris-HC1 pH 7.6, 1.5 mM MgC12, 50 mM NH4C1, 3 mM 2-mercaptoethanol. RNA (25 pmol) and proteins (85 - 100 pmol, see legends to figures and tables) were incubated together at 20 "C for 20 min before [35S]thiostrepton (40 pmol) was added and incubation continued at 20 "C for a further 10 min. One of the following two procedures was then adopted.

(a) The whole incubation mixture was applied to a column (7 x 0.5 cm) of BioGel A-0.5m and eluted with the incubation buffer. Radioactivity in column fractions (100 pl each) was estimated by liquid-scin- tillation spectrometry [3]. Both 23-S and 16-S rRNA are eluted in the void volume on such a column together with any bound thiostrepton and are thereby separated from unbound drug.

(b) To 100 p1 incubation mixture containing RNA, protein(s) and [35S]thiostrepton was added 10 p1 5 (w/v) activated charcoal (Norit) suspended in buffer A. After thorough mixing this was allowed to stand at 20 "C for 10 min, before the charcoal was pelleted by centrifugation at 12000 x g for 5 min. Thiostrepton is quantitatively adsorbed onto charcoal under these conditions unless bound to some component of the incubation mixture. Accordingly, 35S radioactivity in the supernatant represented bound thiostrepton and was estimated by removing samples (75 pl) for liquid-scintillation spectrometry.

In the experiments in Fig. 1, a combination of these two methods was used. In Fig.lA, free [35S]- thiostrepton was removed from an incubation mixture usingNorit as above in(b)priorto gel filtration asin(a). In Fig. 1 B, an incubation mixture containing 23-S RNA and protein L11 in the absence of drug was subjected to gel filtration as in (a) above before the ability of material in column fractions (100 p1 each) to bind [35S]thiostrepton was assayed by the Norit- adsorption technique (b).

RESULTS AND DISCUSSION

The starting point for the present investigation was our observation that 1 M LiCl split proteins from ribosomes of wild-type B. megaterium could restore to total ribosomal RNA the ability to bind [35S]thio- strepton (Table 1) whereas the corresponding split proteins from B. megaterium mutant MJ1, which pos- sesses ribosomes devoid of protein BM-L1 1, were inactive. Subsequently, when purified protein BM-L1 1 was incubated with total ribosomal RNA, a binding site for thiostrepton not present on either the RNA or the protein separately was created (Table 1). Next, we fractionated B. megaterium ribosomal RNA into

Table 1. Binding of [3sS]thiostrepton to RNA-protein contplexes Total ribosomal RNA from B. megaterium wild-type or fractionated ribosomal RNA components (23-S, 1 6 4 and 5-S) from B. megu- rerium or E. coli were incubated with 1 M LiCl split proteins or with purified ribosomal proteins as described under Methods. The amount of RNA used per assay was 25 pmol regardless of whether total rRNA or fractionated components were employed. The inputs of proteins per assay were as follows: 1 M LiCl split proteins 90 pmol equivalents, purified proteins L11 and BM-L1 1 100 pmol. Binding of [35S]thiostrepton to RNA-protein complexes was assayed by the Norit-adsorption technique. Control experiments (data not given) established that none of the RNA or protein preparations used here bound [35S]thiostrepton to any detectable level. Binding ratios were calculated as mol [35S]thiostrepton bound per mol RNA input

RNA input Protein input Binding ratio

B megaterium B megutrrrum total rRNA wild-type 1 M splits 0.67

B megaterrum MJ1 1 M splits 0.09

BM-L11 0.61 ~~~ _ ~ _ ~ _ _ -~

B megairriunz 2 3 3 RNA BM-L11 0.70 16-S RNA BM-LI 1 0 5-S RNA BM-L11 0

E to11 23-S RNA L11 0 77 BM-L11 0 80

B megaterium 23-S RNA BM-LI 1 0 70 L11 0 86

_ _ ~ - ~ - ~ ~ ~ ~ ~ ~

its 23-S, 16-S and 5-S components and observed that only 23-S rRNA participated in thiostrepton-binding (Table 1).

These observations were readily repeated using 23-S rRNA and ribosomal protein L11 from E. coli and also in experiments where 23-S rRNA and protein L11 (or BM-L11) from these two organisms were combined in reciprocal fashion (Table 1). This serves to emphasise the homology between ribosomal pro- teins BM-L11 of B. megaterium and L11 of E. coli and suggests that 23-S rRNA from these organisms must also share homologies of sequence or structure or both.

The ability of 23-S rRNA to form a binding site for [35S]thiostrepton in the presence of protein BM-L1 1 was not affected by the method of preparation of the RNA. This could be achieved by extraction of ribo- somes either with phenol (see Methods) or with LiCl plus urea [8] or with acetic acid [9]. These methods yielded RNA of approximately equal activity in sup- porting drug binding (data not given). Routinely, we have employed RNA prepared from ribosomes by phenol extraction (see Methods).

Protein L11 prepared under non-denaturing con- ditions (i.e. in the absence of urea) binds to 23-S rRNA whereas other denatured preparations of this

J . Thompson, E. Cundliffe, and M. Stark 263

protein do not [lo]. We have observed that protein BM-L11 prepared under either set of conditions was active in forming a thiostrepton-binding site with 23-S RNA and this can also be seen in Table 1 by com- paring the effects of protein L11 ('denatured') and BM-L1 1 ('undenatured'). Usually the non-denatured protein possessed greater activity in this system but we do not unequivocally attribute this to the method of preparation. We would expect some variation in activity from one preparation to another and also with the age of a given preparation. In any event, if thiostrepton stabilises or in any way modifies the complex between 2 3 3 RNA and L11, this might override any normal requirement for a particular state of protein L11 in binding to 23-S rRNA.

When the formation of 23-S RNA-L1 1-thiostrepton complexes was followed over a range of concentrations of Mg2+ (1 -20 mM) and NH: (5-250 mM) and over a range of temperatures (0 - 40 "C), we were sur- prised to find very little variation in the amount of [35S]thiostrepton bound (data not given). Routinely, conditions of 1.5 mM Mg2+, 50 mM NH; and 20°C were employed. Again, we assume that thiostrepton stabilises 23-S RNA-L11 complexes upon binding since we would expect the formation of these com- plexes in the absence of drug to exhibit defined optimal conditions.

Having observed the formation of 23-S RNA-LII- thiostrepton complexes 'using the convenient Norit- adsorption technique to remove unbound drug, we wished to demonstrate their formation and to examine their stability by other means. As shown in Table 2 and Fig. 1 the Norit technique evidently leads to an underestimate of the extent of complex formation. When incubation mixtures were subjected to gel- filtration on BioGel A-0.5m the stoichiometry of binding approximated closely to one molecule of thiostrepton bound per molecule of 23-S RNA present regardless of whether E. coli protein L11 ('denatured') or B. megaterium protein BM-L1 1 ('non-denatured') was employed. It can also be seen from Fig.1B and Table 2 that significant amounts of 23-S RNA-L11 complex formed in the absence of drug were recovered in the void volume after gel filtration where they were detected by their ability to bind [35S]thiostrepton.

From the experiments dkscribed so far we concluded that thiostrepton forms a complex with 23-S rRNA and ribosomal protein L11 (or its homologue, protein BM-L11) and that this complex is comparable in stability with the high-affinity binding of the drug to the intact 50-S ribosomal subunit. Since it is quite exceptional for any antibiotic to bind so tightly to a ribosome or a sub-ribosomal particle, we were re- assured that we were indeed examining the physio- logical binding site for thiostrepton. We therefore decided to investigate whether a fragment of E. coli 23-S rRNA could support the binding of thiostrepton

u 10 20 30

Fraction number OO

Fig. 1. Gel filtration of E . coli 2 3 4 RA'A-LII cornp1ese.s. This ex- periment was carried out as in Table 2. Complexes of 23-S RNA and protein L11 were subjected to gel filtration both in the pres- ence and in the absence of [35S]thiostrepton. (A) Following gel filtration in the presence of the drug, column fractions (100 PI) were assayed directly for radioactivity by liquid-scintillation spec- trometry. (B) Following gel filtration in the absence of drug the ability of each column fraction to bind [35S]thiostrepton was assayed by the Norit-adsorption technique

Table 2. Binding of [35S]thiostrepton to 23-S RNA-protein c'om- plexes assayed by different techniques Complexes of 23-S rRNA from E. coli with either protein L11 from E. coli or BM-L11 from B. rnegarerium were formed as in Table 1. Radioactive thiostrepton was then added and its binding was assayed either by the Norit-adsorption technique or by gel filtration on Bio-Gel A-0.5m as described under Methods. In other experi- ments 23-S RNA and protein were incubated together in the ab- sence of drug and then subjected to gel filtration on BioGel A-0.5m. The ability of column fractions (100 PI) to bind [35S]thiostrepton was then assayed by the Norit-adsorption technique. Binding ratios were calculated as in Table I

Technique employed Protein Binding employed ratio

Gel filtration in the absence L11 0.70 of thiostrepton BM-LI 1 0.96

Gel filtration in the presence L11 0.90 of thiostrepton BM-L11 1 .oo

Norit adsorption L11 0.77 BM-L11 0.80

in the presence of either protein L11 or protein

Incubation of 23-S rRNA with either T1 or pan- creatic ribonuclease totally destroyed its ability sub- sequently to form a thiostrepton-binding site (Table 3A). However when 23-S RNA was allowed

BM-L11.

264 Thiostrepton Binding Site

Table 3. i$'hct of ribonuclense on binding ahility of 23-S RNA E. coli 2 3 3 rRNA (25 pmol) was incubated at 20'-C for 10 min in standard incubation buffer with: (A) 40 pmol [3SS]thiostrepton. (B) 100 pmol of either protein L11 or protein BM-LI 1, (C) [3sS]thiostrepton plus either protein L11 or protein BM-L11. Then either TI ribo- nuclease (100 units) or 0.5 pg pancreatic ribonuclcase (RNase 1) was added and incubation continued at 30-C for 20 min. Following incubation with nuclease, protein L11 or protein BM-LlI was added to (A) and [35S]thiostrepton was added to (B) and these were incubated at 20 'C for a further 10 min. Finally, (A, B and C), each by now 90 pi in total volume, received 10 p1 Norit suspension and binding of [3sS]thiostrepton to RNA-protein complexes was assayed as under Methods

Expt Order of additions

first -~ ~- ~ ~~- ~~

second third

Binding ratio

A. 2 3 3 RNA + [3sS]thiostrepton TI RNase BM-L11 or L11 0.04

B. 2 3 3 RNA + BM-L11 T l RNase [3'S]thiostrepton 0.68 23-S RNA + L11 TI RNase [3'S]thiostrepton 0.23 23-S RNA + BM-L11 RNase I ['5S]thiostrepton 0.53

C 23-S RNA + BM-L11 + ["'Slthiostrepton TI RNase - 0.78 23-S RNA + L11 + [35S]thiostrepton T1 RNase - 0.44 23-S RNA + BM-Ll1 + [3sS]thiostrepton RNase I - 0.72 23-S RNA + L11 + [35S]thiostrepton RNase I - 0.36

23-S RNA + [3SS]thiostrepton RNase I BM-L11 or L11 0.05

23-S RNA + L11 RNase I ["s] thios1repton 0.20

to form a complex with protein L11 (or BM-L11) prior to nuclease digestion, substantial protection of thiostrepton-binding capability was observed (Table 3B). This effect was even more pronounced when thiostrepton was added to the 23-S RNA- protein complexes prior to addition of either nuclease (Table 3 C). Again, this may indicate that thiostrepton stabilises the binding of protein L11 (or BM-Lll) to a particular region of E. coli 23-S rRNA. Ribosomal protein BM-L11 of B. niegaterium was notably more efficient in this system than was ribosomal protein L11 of E. coli and this may again reflect the fact that they were prepared differently.

Finally, we attempted to isolate the complex in- volving an RNA fragment, protein L11 (or BM-Lll) and [35S]thiostrepton following the action of T1 ribo- nuclease (as in Table 3C). This was attempted by gel filtration on Sephadex G-75 (Fig. 2) and by sucrose density-gradient centrifugation (Fig. 3). Protected complexes containing protein BM-L1 1 were readily detected by either method (Fig. 2 and 3) whereas with protein L11 of E. coli only low levels of complexes survived nuclease digestion (data not given). This lat- ter result is understandable given the data in Table 3. Calibration of the gradients with proteins of known molecular weights allowed us to estimate the mean molecular weight of the protected complex as being close to 30000 (Fig. 3). This conclusion is compatible with the position of elution of the complex on Sephadex G-75 (Fig. 2). Given that ribosomal protein BM-L11 has a molecular weight of about 15500 [3] (protein L11 has a molecular weight of about 14900) we conclude that about 50 nucleotides of E. coli 23-S RNA were protected from digestion by T1 ribo- nuclease and remained bound to protein BM-L11 in

i

OO MI/ 10 20 30

Fraction number

Fig. 2. GelJiltration of rhc 23-5' RAJA + Bh"lL.11 conzpl'sfi)lloii.ina digestion with TI ribonuckiuse. The complex of 2 3 3 RNA + BM- L11 + [3'S]thiostrepton was digested with TI ribonuclease (see Table 3C) and, as a control, 23-S RNA was digested with the nuclease in the presence of [35S]thiostrepton prior to addition of protein BM-Ll1 as in Tablc 3A. Each incubation mixture was then applied to a column of Sephadex (3-75 (7 x 0.5 cm) which was eluted with standard incubation buffer. Fractions (100 pl) were collected and radioactivity assayed by liquid-scintillation spectrom- etry. (O--O) RNA-protein complex formed prior to addition of TI ribonuclease; (O----O) RNA digested with T1 ribonuclease prior to the addition of protein BM-L11

the presence of thiostrepton. Failure of protein L11 of E. coli to protect a similar RNA fragment pre- sumably reflects the fact that it was prepared under denaturing conditions and appeared to possess a lower affinity for 23-S rRNA [lo] (also Table 3).

J . Thompson, E. CundliiTe, and M . Stark 265

Fig. 3. Sucrow ck.n.sit~-grudic.nt centriJuga/ion u f the 23-S R N A + BM-LI I coniplex ,follon.ing exposure t o TI riboi~uc/eu.sc~. The complex formed between E. coli 2 3 3 rRNA, protein BM-L11 and [3sS]thiostrepton was digested with T1 ribonuclease as in Fig. 2 and Table 3C. Unbound thiostrepton was removcd by addition of Norit followed by centrifugation (see Methods) and the super- natant was applied to a 5 - 20 "/, (w/v) sucrose density gradient made up in 4.9 ml standard incubation buffer. At the Same time proteins of known molecular weight were layered onto similar gradients for calibration purposes. Centrifugation was carried out for 4 h at 40000 rev./min and 2'C in the Beckman SW 50.1 rotor. After centrifugation the gradients were pumped through an Isco UA-5 ultraviolet absorbance monitor and radioactivity in frac- tions (0.25 ml) from the appropriate gradient was estimated by liquid-scintillation spectrometry. Molecular weight standards : lyso- zyme (14500), deoxyribonuclease I (31 OOO), bovine serum albumin (65000) and E. coli alkaline phosphatase (80000). (0) ["SIThio- strepton; (0) molecular weight

CONCLUSIONS

Thiostrepton binds with high affinity and with 1 : 1 stoichiometry to a complex formed between E. coli 23-S rRNA and ribosomal protein L11 of E. coli or protein BM-L11 of B. meguterium. Such RNA-protein complexes are sufficiently stable to survive gel filtration in the absence of thiostrepton but their resistance to ribonuclease digestion is markedly enhanced in the presence of the drug. We assume that thiostrepton achieves this by stabilising the binding of protein L11 and BM-L11 to a particular region within the 23-S rRNA.

A fragment (or fragments) of 23-S rRNA totalling about 50 nucleotides in length is protected from digestion by T1 ribonuclease in the combined presence

of protein BM-L11 and thiostrepton. Physical studies of protein L11 suggest that it may exist as an elon- gated molecule about 15 nm in length [ l l ] which approximates the length of an RNA fragment of 50 nucleotides. Assuming that proteins L11 and BM- L11 possess similar structures when bound to 2 3 3 rRNA this may indicate that the protected RNA fragment does not contain extensive loop structures. The purification and further characterisation of this RNA fragment are currently being undertaken.

Antibiotics which bind to ribosomes do not usually interact with sufficient affinity with isolated ribosomal components to allow this kind of investigation, al- though recently [12] erythromycin has been shown by equilibrium dialysis to bind to E. c d i ribosomal pro- tein L15. It will be interesting to determine by similar means whether thiostrepton binds weakly to any ribosomal protein other than L11 or its homologue BM-Lll . Such binding might conceivably account for the low-affinity interaction between thiostrepton and ribosomes which lack protein BM-L11 as found in B. meguterium mutants such as MJI .

This work was supported by a research projcct grant (to EC) from the Medical Research Council who also provided a research studentship (for MJRS). We are most grateful to Georg Stoffler for providing ribosomal protein L l l of E. cdr and to Dino Bedlington for technical assistance.

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J . Thompson, E. Cundliffe, and M. Stark, Department of Biochemistry, University of Leicester, University Road, Leicester, Great Britain, LE1 7HR