A plasmid-coded and site-directed mutation in Escherichia coli 23S RNA that confers resistance to erythromycin: implications for the mechanism of action of erythromycin

Biochimie, 69 (1987) 891 -900

891

© Socidt6 de Chimie biologique/Elsevier, Paris

A plasmid-coded and site-directed mutation in Escherichia coli 23S RNA that confers resistance to erythromycin: implications for the mechanism of action of erythromycin Birte VESTER and Roger A. GARRETT Biostructural Chemistry, Kemisk Institut, Aarhus Universitet, DK-8000 /~rhus C, Denmark (Received 19-5-1987, accepted 25-5-1987)

Summary - Primer-directed mutagenesis was employed to introduce a n A2058 --* G transition in plasmidencoded Escherichia coli 23S RNA at a site that has been implicated, indirectly, in erythromycin binding [1]. The mutation raises the growth tolerance of cells from 30 to 300/.tg/ml of erythromycin, and cells grown in the presence of erythromycin contain ribosomes with high levels of mutated 23S RNA. In these cells, wild type 50S subunits 'fall off' the message and are selectively degraded, possibly as a result of an erythromycin-induced conformational change. A fast in vitro poly(U) assay revealed minimal effects of erythromycin on elongation beyond tetrapeptides. We correlated these results with the literature data and concluded that erythromycin acts immediately post-initiation and directly, or indirectly, destabilizes mRNA-bound 70S ribosomes, and prevents their recycling by causing 50S subunit degradation. ribosomal RNA I site-directed mutagenesis I erythromycin resistance

R~sum~ - Une mutation cod~e par un plasmide et dirig~e vers un site dans I'ARN 23S d'Escherichia coli conf~re une r~sistance ~ r~rythromycine: implications dans ie m~canisme d'action de l'~rythromycirie, line mutagdn~se dirigde vers un amorceur a dtd utilisde pour introduire une transition A2oss ~ G dans un A R N 23S d'Escherichia coli codde par un plasmide, ~ un site indirectement impliqud dans l'attachement de l'drythromycine [1]. Cette mutation augmente la toldrunce de croissance des cellules de 30 ~ 300 t~g/ml d'drythromycine et les cellules cultivdes en prdsence d'drythromycine contiennent des ribosomes contenant des proportions dlevdes d ' A R N 23S mutd. Dans ces cellules, les sous-unitds 50S de type sauvage ~ abandonnent ~ le message et sont sdlectivement ddgraddes, possiblement par suite d'un changemerit de conformation induit par l'drythromycine. Un test rapide de poly(U) in vitro rdvble que les effets de l'drythromycine sur l'dlongation sont minimes au-del~l des tdtrapeptides. Nous avons corrdld ces rdsultats avec les donndes de la littdrature et conclu que l'drythromycine agit immddiatement aprbs l'initiation et ddstabilise, directement ou indirectement, ies ribosomes 70S lids ~i I'ARN messager et emp~che qu'ils se recyclent en provoquant la ddgradation des sous-unitds 50S. A R N ribosomal I mutagdn~se dirigde vers un site I rdsistance ~ l'drythromycine

Introduction Several antibiotics that inhibit specific ribosomal functions act at the level of ribosomal RNA as revealed by both methylation and mutation of RNA

which confer antibiotic resistance, and by direct cross-linking of antibiotics to RNA (reviewed in [2]). Thus, elucidating antibiotic binding sites is crucial for defining the functional roles of ribosomal RNA.

892

B. Vester and R.A. Garrett mutagenesis and expressed the mutant RNA on a multicopy plasmid in order to investigate its effect on ribosomal activity in the presence and the absence of erythromycin.

The macrolide erythromycin binds to 50S subunits and 70S ribosomes at a 1:1 molar ratio but poorly to polysomes unless nascent peptides are prereleased by puromycin treatment [3, 4]. There are indications, that the antibiotic acts at the translocation step of protein biosynthesis [5, 6] while an alternative view suggests that it acts immediately post-initiation at a critical, but undefined, stage of the ribosome cycle ([3] and references therein). Current evidence supports a primary binding site for erythromycin on 23S RNA. Streptomyces erythraeus, the producer of erythromycin, is N6-dimethylated at the position corresponding to A-2058 of E. coli 23S RNA and this accounts for the host resistance [1]. Mutations have also been observed at equivalent, or neighboring positions, in the RNA secondary structure in yeast mitochondrial R N A s A2058 --* G [7,] C2611 ~ G [8] and op, multicopy plasmids carrying E. coli 23S RNA genes A205s -* U [9], C2611 --* G [10]. They lie in the

Materials and methods Bacterial strains, vectors and cloning E. coli strains HBI01 [13] and GM48 [14] were used as plasmid hosts. The growth medium contained 10 g of tryptone (Difco), 5 g of yeast extract (Difco) and 5 g of NaCI per liter. Ampicillin and erythromycin were from Sigma. Plasmid pKK3535 containing the rrnB rRNA operon [15] was used for cloning. Expression vectors M13mp9 and mp8 [16] were used for cloning, site-directed mutagenesis and sequencing. Plasmids and replicative forms of M13 clones were prepared by standard procedures [13]; single-stranded phage DNA was purified essentially as described in [17]. Digestion with restriction enzymes (Amersham), DNA ligation with T4 ligase (Amersham) and DNA transformation were performed as described in [13]. The insertion of fragments into the MI3 vectoi's was checked by the capacity of clones to generate 'figure-eigl~ts' [18]. Construction of expression plasmids pDV and pDVE is described under Results.

generally conserved central loop of domain V which has been implicated in peptidy] transfer [2] (see Fig. 1). In the present study, we have substituted A205s in E. coli 23S R N A with a G by site-directed

U "......,.... ..... -....

/ /

/

U ,G

I

A- 1252 (domoinIT)

A

A/2°2°

""\ G A A \ m2AMLSr U GU • G \\'~ UMLS r

C

Uc,°.%~A'c'oU •

G -- C C -- G

(~)

Ery~___

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(~)_ ,.G,r_-,

,~\\\ zu~u "¢~ G,-, 2/.50 "~X,,.X\ X~.'(~',,A ,.'-z, (,_A;I:O,_/

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c ~ A ?.-,.A,-" "

G ~ 2o~o U

~-

A

I I I GGGU

" -

I I I I I I I GCCGUCU C(~)~ ,A,,U~ C 2s2o U

Er, r

"

I I..

G Ery

C O O ) u A

G

C U--2600

C 2600

(~i.G', ,C

A,~,U U\ \r'. \

,G,

•

,,'¢,- c

6-" "c u-U l ~

..G,

/-

BP-PhetRNA crosslink

";~ n

"'¢'~

25ao---LU., • G C -- ,~.',

Fig. 1. Secondary structural model of the central part of domain V of E. coli 23S RNA [2]. Conserved nucleotides are shown by dotted circles. Dashed lines indicate putative tertiary interactions [11]. We indicate the site of dimethylation [1] and the mutations that confer erythrornycin resistance: A --, U [9], A --, G [7], G --, A [10], and C --, G [8]. MLS: macrolide, lincomycin and streptogramin B. An affinity labeling site for benzophenone-derivatized Phe-tRNA [12] is indicated.

Erythromycin resistance and 23S ribosomal RNA The mutation was confirmed by cloning the 507 bp

Sau3A fragment from plasmid pDVE (see Results) into M13mp9 and sequencing through the mutated position [19]. rRNA was sequenced using deoxyoligonucleotide primers and reverse transcriptase (Life Sciences) [20].

Primer-directed mutagenesis A 15-mer dGGTCTTCCCGTCFTG (the mutating nucleotide is underlined) was synthesized by the solid-phase phosphotriester method [21]. Site-directed mutagenesis was performed on the single-stranded M 13 clones by annealing the mutagenic primer and extending it by adding dNTP's and Klenow fragment (Amersham) [22]. cc-DNA was enriched by agaxose gel electrophoresis [13]. Dot- blot hybridization was performed by spotting supernatants of M 13 extracts on Gene Screen membranes (NEN). DNA was denatured in 0.2 M NaOH and 0.6 M NaCI, neutralized in 0.5 M Tris-HCl, pH 7.5, 1.5 M NaCI and rinsed in 0.3 M NaCI, 0.03 M sodium citrate and 2, mM EDTA, pH 7.2, and the membrane was baked for 3 h at 80°C. Prehybridization and hybridization steps were performed as described earlier [22]. Plaques were screened and the replicative forms of potential mutants were isolated. The mutated 23S rDNA fragment was excised from the MI3 phage by Sail and EcoRI for plasmid construction.

Preparation of ribosomes and poly(U)-directed polyPhe synthesis Cultures of HBI01/pDV and HBI01/pDVE were grown to 0.7 A650 (the latter in the presence of 200/.tg/ml of erythromycin) and ribosomes were isolated according to Jelenc [23] whereby salt-washed 70S ribosomes were separated by gel filtration on Sephacryl S-200 (Pharmacia) and stored at -80°C in 'polymix' buffer (5 mM MgCI2, 0.5 mM CaC12, 8 mM putrescine, 1 mM spermidine, 5 mM K3PO4, 5 mM NH4CI, 95 mM KCI, l mM dithioerythreitol, pH 7.5). Ribosome and subunit ratios were measured as follows: 33/.tg of the ribosome sample were diluted to 50 ~zl with 30 mM Tris-HCl, pH 7.5, 10 mM MgCI2, 100 mM NH4CI and centrifuged in a Beckman SW 60 Ti rotor for 12 h at 20000 rpm at 5°C in a 5-20°7o (w/v) sucrose density gradient containing the same buffer. The solution was eluted and analyzed at 260 nm in a Gilson Spectrochrom M spectrophotometer. PolyPhe synthesis was performed in vitro essentially as described by Wagner et al. [24]. Initially, ribosome and factor mixtures were combined after preincubating for 10 min at 37°C. The former contained ribosomes, poly(U) and [3H]NAcPhe-tRNAPhe in 'polymix' buffer [24] which complexed during preincubation. The latter contained total tRNA, Phe-tRNA synthetase, [~4C]Phe, EF-Tu, EF-Ts, EF-G, ATP, phosphoenolpyruvate, pyruvate kinase, and myokinase in' polymix' buffer; enzymes were added at concentrations in excess of those required for the maximum elongation rate [24]. In some experiments, EF-G concentrations were limited in order to decrease the elongation rate and, thereby, emphasize the start of elongation, The fraction of active ribosomes was determined by incorporating [3H]NAcPhe at the N-

893

terminii of polyPhe. Only peptides longer than 4 amino acids were precipitated by trichloroacetic acid and their yield was measured by total [14ClPhe incorporation.

Results This section describes the construction of the mutant plasmid, its characterization and how it affects erythromycin resistance both in vivo and in vitro.

Construction o f the m u t a n t plasmid An A -~ G transition at position 2058 of the plasmid-cod~d 23S RNA gene was produced as outlined in Fig. 2. Briefly, a SalI-BclI fragment was excised from the 23S R N A gene in pKK3535 [15] and cloned into an M13mp9 vector cut with SalI and BamHI. The mutagenic primer was hybridized to the ssM 13 DNA and extended by adding dNTPs and Klenow fragment. After ligation, closed circular DNA was isolated and transformed; 96 plaques were screened for mutation and two were positive. A SalI-EcoRI fragment was excised from one of these and 3-point ligated with BamHI-Sall and EcoRI-BamHI fragments from pKK3535 to generate pBVE containing the mutated fragment cloned back into the rRNA operon (Fig. 2). The choice of fragment for cloning was limited due to a lack of appropriate r~.striction enzyme sites in pKK3535. The prcJcedure was also complicated by the requirement for a ~.onmethylating (dam-) host for the Bcll enzyme and the need for a partial restriction enzyme digest of the piasmid prior to the 3-point iigation. Plasmid pDV was constructed from pKK3535 by deleting a BclI-EcoRI fragment, as described above, followed by deletion of an NaeI fragment which contained a PstI site (Fig. 2); the remaining PstI site in the A m p r gene was useful for later clonings. A small PvuII-Pst! fragment from pBVE was combined with a larger PvuII-PstI fragment from pDV to construct the final mutated pDVE plasmid (Fig. 2). The control plasmid pDV and pDVE differed only at the mutation site.

E f f e c t s o f the mutation in vivo The mutated multicopy plasmid pDVE (A205s --, G) and plasmid pDV were transformed into HB101 cells. The gene dosage from similar plasmids produces about half of the cell's r R N A [25] but does not lead to overproduction of rRNA because plasmid- and chromosome-coded rRNA genes are co-regulated [26].

894

B. Vester and R . A . Garrett

RNA was incorporated into functionally active ribosomes and, second, that the A --, G change induced resistance to erythromycin. The effect of erythromycin on the cellular content of mutant ribosomes was investigated. Ribosomes were prepared from cells containing pDVE grown at 200/zg/ml of erythromycin and, as controls, from pDVE and pDV-containing cells grown in the absence of the antibiotic, rRNA was isolated and sequenced through the appropriate region of 23S RNA with reverse transcriptase, to reveal a mixture of wild type A and mutant G at position 2058, as depicted in Fig. 3. Densitometry of the bands in this region are shown in Fig. 4, and normalization of their peaks demonstrated that ribosomes were depleted in wild type 23S RNA by 29°70 in the presence of 200 #g/ml of erythromycin. Examination of the ribosome/subunit ratios in the presence and the absence of the antibiotic provided a rationale for this result. Ribosomes from pDVE-containing cells were eluted from Sephacryl S-200 columns and analyzed on a sucrose gradient. The result shown in Fig. 5 demonstrates that 30S subunits accumulated in the presence of erythromycin. Densitometry of the peaks revealed

Table I shows doubling times for cells containing both control (pDV) and mutated (pDVE) plasmids. The latter grew about 1007oslower in the absence of erythromycin and, therefore, the mutation has a small deleterious effect on ribosome function. However, pDVE-cells exhibited high antibiotic resistance and grew at 300/zg/ml of erythromycin (doubling time not determined), whereas pDVcontaining cells stopped growing by 50 btg/rnl. This establishes first that the plasmid-coded mutant 23S Table I. Doubling times (min) for E. coli cells transformed with plasmids. Plasmid

pDVE pDV

Erythromycin ~ g / m l ) 0

50

47 43

72 .

lO0

150

250

84

90 .

126

.

.

Cultures grown overnight with 50/zg/ml of ampicillin and increasing amounts of erythromycin. They were diluted to about 0.02 ODs4o and then 0.2 ml of each was transferred to a titerbox. Growth was continued at 37°C and measured for at least 3 doubling times.

P

Bcl I

Born HI

- -

co RI

-

Pst I

,Pst I

@ Fig. 2. Construction of mutant plasmid pDVE. Restriction sites used for fragmentation are indicated. In the numbering system for pKK3535 [15] the sites are: BamHI (1), Bcll (6954), EcoRI (7880), IVael (634; 10961), Pstl (8632), Pvull (5260), Sail (4842) and Sau3A (5383; 5890). The mutated position is denoted by an arrow head. Experimental details are provided in the text.

Erythromycin resistance and 23S ribosomal RNA that 50S:30S subunit ratios correspond to approximately 1.7 in the absence of erythromycin and 0.3 in its presence. Since we showed in Fig. 4 that this ribosome preparation contained less unmutated 23S RNA, we could conclude that the wild type 50S subunits had been selectively degraded.

895

A -Ery

0.8-

Effects o f the mutation in vitro The effect of erythromycin on mutated and wild type ribosomes was investigated, using an in vitro poly(U) assay, in which polyPhe is synthesized at a rate and accuracy comparable to in vivo protein synthesis in the presence of elongation factors [24]. The assay yields both the degree of incorporation of phenylalanine and the number of elongating ribosomes. It is possible to establish, therefore, whether reduced synthesis reflects a drecreased rate of synthesis or fewer elongating ribosomes. Ribosomes were prepared from HB101/pDV and HB101/pDVE cells and their activities in the poly(U) assay were measured at increasing erythromycin concentrations. All assays included a 10 min preincubation of the ribosome and factor mixtures at 37°C in order to charge the tRNA and to complex NAcPhe-tRNA and poly(U) to ribosomes. Elongation commenced immediately after combining the mixtures. Addition of the antibiotic did not affect the elongation rate either for pDV ribosomes, or for pDVE ribosomes which contained both wild type and mutant RNA. However, when limiting amounts of EF-G and a short incubation time were used to slow down

0.4-

0.0B +Ery 0.8

0.4

0.0 0

G-2046

mutant - Ery G

+ Ery A

G

wildtype A

G

A

T

C

A A G A C G G A-2058 A

C-2064

Fig. 3. Autoradiogram showing the sequence through the mutated position in the wild type (A-2058). The reverse transcriptase procedure was used for sequencing (see Materials and Methods). A and G tracks are shown for 23S RNA isolated from mutated ribosomes grown in the absence and the presence of erythromycin. The mutated position is indicated by an arrow.

I

cm

2

Fig. 4. Densitometer traces of the autoradiogram shown in Fig. 3 showing the relative decrease in the unmutated A-2058 in the presence of 200/,tg/ml of erythromycin. The autoradiograms were scanned on a Shimadzu CS-930 scanner which integrated the peak areas. The areas were normalized by calculating the ratios of the 6 peaks adjacent to the mutated position. The average ratio ( - E r y / + Ery) was 1.32 + 0.10. The value for band A-2058 was 1.87 which corresponds to a decrease of 29°70 in the peak area of the wild type band in the presence of erythromycin.

elongation, and emphasize its start phase, there were changes in the number of elongating ribosomes. Results obtained for increasing erythromycin concentrations (0-1100/zg/ml) are depicted in Fig. 6. The number of elongating pDV and pDVE ribosomes decreased and increased, respectively (Fig. 6A), while the elongation rates of both remained constant (Fig. 6B).

896

B. Vester and R . A . Garrett

due to mutated ribosomes assembling in the pDVEcontaining cells. This result confirms the involvement of this adenosine in erythromycin binding in eubacteria and some mitochondria. The mechanism of erythromycin action was investigated by comparing its effect on wild type ribosomes and on those containing mutated 23S RNA in a poly(U) assay that detects tetrapeptides and longer. There resulted a decrease in the number of elongating wild type ribosomes and an increase

-Ery

1

•

A

pDVE

g

Eo .o "t-

J

70S 50S 30S

f

70S 505 30S

>=

O O

(.}

o

O

O

pDV

8

ut (Is

Fig. 5. Elution profiles for sucrose gradients of the ribosome preparations (see Materials and Methods) from pDVE-containing cells grown in the absence and the presence of 200/,tg/ml of erythromycin. Peak areas were determined by planimetry.

(x 7

6

I 200

0

Various parameters were varied during the in vitro assay to establish whether the effects could be altered. For example, ribosomes were preincubated with erythromycin prior to tRNA or poly(U) binding, and limiting amounts of EF-Tu and EF-Ts were used, but no further changes were detected. Only at excessively high concentrations of erythromycin (15000 ~ag/ml) was polyPhe synthesis totally inhibited, but this was probably due to the antibiotic acting at secondary sites. Also, the possibility that the A ~ G mutation affected the translational error level was investigated using the poly(U) assay to obtain error frequencies based on the incorporation of Lys relative to Phe [24] but no effect was observed. We, conclude from these results first, that erythromycin affects mutated and unmutated ribosomes differently an,l second, that elongation of poly(U)-directed polyPhe synthesis is almost unaffected by erythromycin.

Discussion Inducing a n A2058 -~ G change in E. coil 23S RNA conferred erythromycin resistance upon the cells

I 400

I 600

I 800

r

I 1000

$tg/ml erythromycin

8 .8 °

B

sI &

Oo~Q

O •

o

8

E

3

3-

E 00_

0

• p DVE

2-

L..

o pDV

3

. c_

I1

0

I 200

I /.00

I 600

I 800

I 1000

Fg/ml erythromycin

Fig. 6. The effect of increasing amounts of erythromycin in the poly(U) assay on the number of elongating ribosomes from pDVand pDVE-containing cells (A) and on the elongation rate (B). The assay was performed over a short time period (7 s) and at low concentrations of EF-G to emphasize the start of elongation. Experimental details are given under Materials and Methods.

Erythromycin resistance and 23S ribosomal RNA in the n u m b e r o f elongating m u t a n t ribosomes (Fig. 6A) and no change in the elongation rate (Fig. 6B). The induced changes were too small to a c c o u n t for t h e cell g r o w t h i n h i b i t i o n by erythromycin (Table I). Moreover, no effects were observed in the presence of optimal a m o u n t s of EF-G. Therefore, we conclude that erythrornycin acts at the start o f elongation (pretetrapeptide formation) or earlier.

897

Various roles have been attributed to it: that it blocks translocation [27] ; prevents correct positioning o f peptidyl-tRNA [28] ; releases peptidyl-tRNA [29], blocks elongation beyond a certain peptide length [5], or that it blocks some critical step in the ribosome cycle immediately post-initiation [3]. These roles are not necessarily incompatible but they are difficult to reconcile. This difficulty is emphasized, for example, by the data of M a o and R o b i s h a w [30] w h o s t u d i e d the effect o f erythromycin on the reaction of puromycin with the various peptidyl moieties o f P-site bound t R N A on 50S subunits, in the presence o f methanol; transfer o f a b o u t half of the peptidyl substrates was reduced and transfer of the rest was stimulated. Further results are summarized in Table II for the reaction o f p u r o m y c i n with various peptidyl tRNAs (and analogues) on 70S ribosomes, in the absence of

Earlier ~tudies on the mechanism o f erythromycin action Literature d a t a indicates that e r y t h r o m y c i n acts post-initiation because while it does not affect the binding o f f M e t - t R N A to R17 R N A - e n c o d e d ribosomes ([3] a n d references therein), it does affect translocation either directly or indirectly [5, 6].

II. Effect of the composition of the elongating peptide on the capacity ef erythromycin to inhibit transfer of the peptide to puromycin on 70S ribosomes.

Table

Inhibition

Ery a °70b

Ref.

No change

Ery

°70

Ref.

Stimulation

PolyLys-tRNA

1 1 5 10 10 80

[31] [32] [3 l] [32] [33]c [34]

fMet-tRNA AcPhe-tRNA

10 10 80 10 80 80

102 101 106 92 102 103

[35] [33]c [34] [33]c [34]

AcPhe-tRNA

Phe2-tRNA

20 15 0 0 25 22

AcPhe2-tRNA GlyPhe-tRNA LeuPhe-tRNA

Ery °70

1 1 AcPhe-CACCA 1 AcLeu-CACCA 1 ValGiyPhe-tRNA 80

150 130 132 126 117

Ref. [31] [32] [32] [32] [34]

[34]

a Ery: erythromycin (/zM); molecular weight = 734. b The percentages express the degree of transfer of the aminoacyl residue to puromycin in the presence of erythromycin relative to transfer with no erythromycin. c Denotes experiments with the EF-G-dependem puromycin reactions. Discrepancies between the data for AcPhe-tRNA may reflect differences in preincubation and buffer conditions.

Table

III. Comparison of the effects of erythromycin on protein synthesis in different assays.

E. coli A. IF-free polysomes [3] Natural mRNA Free ribosomes Initiation factors Di- and tripeptides formed Erythromycin conc. (M) Assay time Inhibition of synthesis

B. subtilis B. RI7 mRNA polysomes [3]

C. Initiating free ribosomes [3]

D. Poly(U) [present data]

E. Poly(U) [36]

+

+

+

-

-

10-6 10 -4 6 min 6 min 10070 30°70

+ + +

+ + 10-3 5-20 sa < 15°70

+ + + 4 × 10-5 -30 min 45°70

10-6 10-4 6 min 6 min 5070 25°70

10 -6

30 rain 70%

The table shows differences between the assays and their responses to erythromycin. + and - indicate the presence and absence, respectively, of a component in the assay. a Preincubated with erythromycin for 10 min.

898

B. Vester and R . A . Garrett

methanol. The Eata suggest that factors, such as

composition (see also [36]) and length of the growing peptide, affect the antibiotic action. Most, of these data, including our own, are compatible with the conclusion of Contreras and V~quez [4], that the main effect of erythromycin is to sterically hinder nascent peptides, 2 - 5 amino acids in length. The data of Tai et al. [3] and those of our poly(U) assay are also compatible with erythromycin acting at an earlier stage of elongation. An erythromycin resistant mutant has also been isolated by Pardo and Rosset [37] that is probably altered in the 30S subunit and affects initiation. Therefore, we examined the literature for data deriving from other protein synthesizing systems [3, 36] in an attempt to examine this possibility. The results in columns A and B of Table III on elongating polysomes, and our data with poly(U) (column D), show that minimal effects occur during elongation. Only when free 70S ribosomes and initiation factors were employed (column C) was there strong inhibition. Thus the main effect must occur pre-elongation. Furthermore, our results with poly(U) in the rapid assay gave little inhibition, whereas the slow assay with B. subtilis (column E) gave strong inhibition. This suggests that the kinetics are crucial and that in our more natural elongating system, a much larger number of ribosomes overcome a block at the start of elongation. E r y t h r o m y c i n b i n d i n g site

The 3 site mutations that confer erythromycin resistance (Fig. 1) are neighbors in the secondary structure, and G-2057 and C-2611 are base-paired. Together they appear to define the RNA binding site of erythromycin. This inference is supported by the accessibility of nucleotides A-2058 to G-2061 to dimethyl sulfate and diethyl pyrocarbonate in the 50S subunit (J. Egebjerg and H. Leffers, personal communication). Furthermore, it is reinforced by the phylogenetic evidence presented in Table IV which shows that only organisms with the composition equivalent to G-2057, A-2058 and C-2611 are erythromycin-sensitive. A possible m e c h a n i s m o f action o f erythromycin

Only mutated ribosomes function in the presence of erythromycin (Table I) and therefore, wild type ribosomes are preferentially excluded from protein biosynthesis. Our results show that the wild type 50S subunits, but not 30S subunits, are preferentially degraded in the presence of erythromycin

IV. Phylogenetic comparison of the three nucleotides that are implicated in the action of erythromycin. Table

Organism

Positions in E. coli Erythromycin 23S RNA tolerance 2057 2058 2611

Eukaryotes

(cytoplasmic ribosomes) P. polycephalum D. discoideum S. cerevisiae O. sativa X. iaevis mouse

A A A A A A

G G G G G G

U U U U

A A C C C

U G G G G

U U G G G

C

G

G

G

A

C

G G G

A A A

C C C

G G

A A

C C

generally sensiti~e[40]

G G A A A

A A G G G

C C U U U

sensitive[40] sensitive[7] (resistant)[81 (resistant)[8] resistant[41]

G G A G G

G U A A G

C C C G C

resistant[ours] resistant[9] resistant[10] resistant[8] resistant[71

U -

generally resistant[38]

Archaebacteria D. T. H. M. H. M.

mobilis tenax halobium vannielii morrhuae thermoautotrophicum

generally resistant[39]

Eubacteria E. coli B. stearothermophilus B. subtilis A. nidulans

generally sensitive [38]

Chloroplasts N. tabacum Z. mays R A d!. ..! IVll I L U I . , I I K , P l I U d [ ll,'ll

P S. A. H. R.

primaurelia cerevisiae nidulans sapiens ratus

E. coli mutants

Yeast mitochondria mutants

Literature references for the sequencesare listedin [42]and [11]. Sequence alignments are derived from Leffers et aL [11] and unpublished results.

(Fig. 5). Thus the 50S subunits fall off the mRNA; the 30S subunits will either fall off concurrently or subsequently be chased off by ensuing ribosomes. Erythromycin must, also, render 50S subunits susceptible to degrading enzymes.

Erythromycin resistance and 23S ribosomal R N A

Erythromycin therefore has two effects. First, to effect' fall off' of ribosomes from the m R N A and, second, to cause degradation of 50S subunits. All of the indications are that the former occurs immediately post-initiation, or within the first 3 cycles of elongation. Selective degradation of the 50S subunits, possibly resulting from an erythromycininduced conformational change, then prevents reformation of 70S ribosomes. Finally, we have some insight into how erythromycin might effect a conformational change in the 50S subunit. Resistance has been correlated with the deletion of nucleotides 1219-1230 from domain II of E. coli 23S RNA by Douthwaite et al. [43]. They inferred that the resistance resulted from a perturbation of an interaction between domains II and V. Recent putative tertiary basepairing between A-2058 (domain V) and U-2016, and the adjacent U-2015 with A-1252 (domain II) (Fig. 1) provided a possible basis for such an interaction [11]. Each of the four mutations (Table IV) and dimethylation of A-2058 produce erythromycin-resistance, and also weaken or disrupt the G-205 7/C-2611 or the A-2058/U-2016 pairings. There is a further potential correlation between the two domains. Mutants resistant to high levels of erythromycin exhibited altered phenotypes in either L4 or L22 ([37] and references therein) both of which associate with the 13S RNA fragment constituting domains I and II [44, 45]. Moreover, reconstitution experiments demonstrated that adding L 16 to protein-depleted cores was required for ,~,-~h,-~, ~O ,l l;l .K,~at l:l -l ~, . r~,:~ ~,A j i, a a z v A .a J.t . y.~ , l ;. l.l ["ml'OJ, and LI6 associates with domain IV or V [44, 47].

Acknowledgments We are very grateful to Professor C.G. Ku,'land and his colleagues at the Biomedical Centre, Uppsala University for their generous help and stimulating discussions during the performing of protein synthesis assays. B.V.'s visit was supported by an EMBO travel grant. We thank J6rgen Kjems for helping with the preparation of the mutagenic primer. The plasmid pKK3535 was provided by Harry F. Noller. Steve Douthwaite kindly performed the NaeI-NaeI deletion on plasmid pBV. Arne Lindahl ana Lisbeth Heilesen helped with the manuscript. B.V. received a licentiate grant from the Carlsberg Foundation. The research was supported by the Danish Medical Science Research Council. We would like to remember to David Vfizquez for his sense of fun, for his inspiration and, especially, for his excellent science.

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