An in Vitro Study of the Translational Attenuation Model of ermC

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Val. 262, No. 4, Issue of February 5, pp. 1756-1765,1987 Printed in U.S.A.

An in Vitro Study of the Translational Attenuation Model of ermC Regulation*

(Received for publication, July 7, 1986)

Chittampalli S. Narayanan and David Dubnau From the Department of Microbwlogy, The Public Health Research Institute of the City of New York, Zm., New York, New York 10016

We have used a Bacillus subtilis in vitro translation system to test the translational attenuation model for ermC regulation. The ermC gene product is known to methylate rRNA, rendering ribosomes unable to bind this antibiotic. We have shown that the induction of ermC methylase in vitro is post-transcriptional and specific for the macrolides erythromycin and oleandomycin. Erythromycin has no significant effect on the stability of the ermC transcript in vitro, and hence the post-transcriptional induction of methylase under these conditions occurs by stimulation of translation. The induction effect requires ribosomes able to bind erythromycin. By adding small proportions of unmethylated to a methylated extract in the presence of erythromycin, methylase synthesis could be induced. Con- versely, when small amounts of methylated extracts were mixed with unmethylated extracts, methylase synthesis could be maintained at elevated levels in the presence of a high concentration of erythromycin. These effects were specific for the inducible ermC, were not observed with a constitutive variant, and could be explained satisfactorily by the translational attenuation model. The roles of three segments of the ermC leader in regulation were explored by probing with appropriate complementary synthetic oligodeox- ynucleotides. The induction effect of erythromycin was mimicked by using an oligonucleotide that could free the ribosome binding site for methylase.

The regulation of gene expression in prokaryotes can occur at the transcriptional or translational level. Post-transcriptional control of gene expression is exerted in several ways. Messenger stability has been shown to be crucial in a few cases, such as the expression of the phage X integrase gene (1). Translational repression by proteins also occurs, as in the syntheses of the coat protein of MS, phage (2) and the T, gene 32 protein (3). Repression by RNA complementary to mRNA occurs in the regulation of TNlO transposition (4) copy number control in certain plasmids (5) and in the regulation of ompF (6). In several other systems, the control of gene expression depends on the mobility of ribosomes on the message. In MS, phage, translation of the replicase message is activated by the translation of the upstream coat protein mRNA (2). In the ermC system of gene regulation, an antibiotic-dependent stalling of ribosomes augments translation (7, 8).

* This work was supported by United States Public Health Service Grants AI-17472 and GM-37137 (awarded to D. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisenent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ermC is carried on the Staphylococcus aureus plasmidpE194 (9) and was later introduced into Bacillus subtilis (10). It confers resistance to the MLS’ antibiotics. The ermC gene product is a 29,000-Da rRNA methylase (11) which dimeth- ylates adenine 2058 of 23 S RNA (12), reducing the ribosomal affinity for MLS antibiotics (13). ermC contains two ribosome binding sites separated by 115 bases within a leader sequence (Fig. 1). The first such site (SD1) precedes a small ORF with sufficient information to encode a 19-amino acid peptide (14, 15). Secondary structure analysis of the leader region of the ermC transcript indicates that the ribosome binding site for the leader peptide message is available for ribosome interaction and binds to ribosomes and that the ribosome binding site for the methylase message is sequestered by base pairing (16). The synthesis of methylase can be induced post-transcriptionally by low concentrations of erythromycin, a protein synthesis inhibitor (13). To explain this effect, the translational attenuation model was proposed (14, 15). According to the model, in the presence of inducer erythromycin-bound ribosomes translate the leader peptide and stall near the end of the leader peptide. This frees the ribosome binding site for the methylase (SD2), enabling translation by erythromycin- free ribosomes. Several in vivo studies employing point, deletion, and substitution mutants of the gene, as well as the structure probing studies mentioned above, have provided strong support for the model (16-19). In this paper we have further tested several aspects of the model, using a B. subtilis i n vitro translation system.

EXPERIMENTAL PROCEDURES AND RESULTS*

To test the fidelity of the B. subtilis translation system and its usefulness for studying ermC regulation, several well characterized templates were used. Fig. 2 shows the results of translation in an S-30 extract with and without added DNA. Lanes 1-3 contain blanks with rifampicin and increasing amounts of S-30, and lanes 4-6 are blanks without rifampicin. The differences between the two blanks are due to the presence of chromosomal DNA in the extract. Lanes 7-10 present total translation products of pE194 with increasing amounts of DNA. The plasmid-coded proteins are easily distinguisha-

The abbreviations used are: MLS, macrolide-lincosamide-strep- togramin B; Cy, clindamycin; Em, erythromycin; Om, oleandomycin; ORF, open reading frame; Rif, rifampicin; SD, Shine-Dalgarno site; TEAB, triethylammonium bicarbonate; Ty, tylosin; Et, ethyl.

* Portions of this paper (including “Experimental Procedures,” part of “Results,” part of “Discussion,” and Fig. 1) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Be- thesda, MD 20814. Request Document No. 86M-2288, cite the au- thors, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

1756

In Vitro Study of ermC Translational Attenuation 1757

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FIG. 1. Secondary structure of the ermC leader transcript. Regions complementary to the 3 oligonucleotide probes are shown. The sequences of the oligonucleotides are given, and the positions of SD1 and SD2 are indicated. The UAA and SD2 mutations are marked.

ble from the background. This is also obvious with $29 DNA, in which case the translation products are almost completely devoid of background. The 429 genome codes for the synthesis of three major proteins of 22.4,13.9, and 10.5 kDa (20). These are all visible in Fig. 2, demonstrating the fidelity of the system.

Since pE194 DNA was used extensively, the proteins specified by pE194 were investigated in some detail in order to characterize the system. pE194 linearized at various sites and appropriate DNA fragments from the plasmid were used in

FIG. 2. Translation of pE194 DNA. Translation was carried out as described under “Experimental Procedures” with 15 mM M$+. Lanes 1-6 contained no external DNA. Lanes 1-3 contained 3 p~ rifampicin (added prior to adding S-30). The volumes of S-30 used in different lanes are: 1 p1 for lanes 1 and 4; 2.5 p1 for lanes 2,5, 7-10; 5 pl for lanes 3 and 6. Lanes 7-10 contained, respectively, 0.5,2,5, and 10 pg of plasmid pE194 DNA. Lane M shows the 629 translation products. Molecular masses are given in kDa (20).

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FIG. 3. Map of pE194. Restriction sites referred to in the text are marked, as well as three open reading frames. ORF B corresponds to ermC, and ORF C is in the replication region. ORF A encodes a site-specific recombinase.3 E l 4 denote the major polypeptides de- tected in the present study.

identifying the plasmid-coded proteins. Both coupled and uncoupled translation systems were used, the latter with low and high concentrations of E. coli RNA polymerase. Fig. 3 presents the pE194 map with the restriction sites used in this study and ORFs marked. Fig. 4 shows the translation products with uncut and cut pE194. Proteins El, E2, E3, and E4 are marked. (Protein E4 is not the same as that described previ- ously in minicell studies (21).) With low concentrations of RNA polymerase, E4 is preferentially transcribed. Plasmids cut with BclI, HueIII, and HpaI fail to show proteins E2 or E3. These sites are within ORF B. This region has been well characterized and encodes the ermC determinant of pE194 (14, 15). It is clear from Fig. 4 that the ermC methylase (E3) is induced when 0.05 pg/ml erythromycin is included in the reaction mix. The SstI site is within the ermC promoter, and

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Vitro Study of ermC Translational Attenuation H p a l l Psi1 I Sphl I I Sstl I

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FIG. 4. Translation of restriction enzyme-cleaved pE194 DNA. The translations were carried out as described under “Experimental Procedures” (with 15 mM M e ) , using plasmid DNA digested with various restriction enzymes. Lanes 1 and 2, coupled translation. Lanes 3-6, uncoupled translation in the presence of rifampicin, using transcripts obtained by transcribing various DNA samples with 0.5 pg of E. coli RNA polymerase (lanes 3 and 4 ) , or 2.0 pg of E. coli RNA polymerase (lanes 5 and 6). Lanes 1, 3, and 5 contained no erythromycin, and lanes 2, 4 , and 6 contained 0.05 pg/ml erythromycin. Lane 5 in the uncut panel inadvertently received an excess of erythromycin and was inhibited. pE194 proteins El-E4 are marked. E4 is not the same as described elsewhere (21). 629 proteins are marked in kDa and are displayed in lane M.

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FIG. 5. Translation of restriction fragments of pE194 DNA. HpaII-ClaI A fragment (panel a) , HpaII-ClaI B fragment (panel b), HpaII-ClaI C fragment (panel c), and CfoI B fragment (panel d). Lane 2 of a, c, and d is a transcription blank containing DNA fragments but no E. coli RNA polymerase. These fragments were translated in the presence of rifampicin. Lane 3 shows a coupled translation. Lane 1 (and lane 2 of b) show uncoupled translation using the appropriate DNA fragment transcribed with 5 pg of RNA polymerase and then processed and translated as described under “Experimental Proce- dures,” using 15 mM M$+. Panel b, lane 1, contained no erythromycin, and lane 2 contained 0.05 pg/ml erythromycin. B, blank with rifampicin; B ‘, blank without rifampicin; M, 629 proteins.

cleavage with this enzyme greatly reduces translation of E2 and E3. E2 and E3 can also be synthesized using the HpaII- ClaI fragment containing ORF B as template (Fig. 5b). E2 will be further discussed below.

Protein E l is made in small amounts and is difficult to identify in some of the lanes. The ORF coding for this protein was confirmed by translating the HpaIIB fragment of pE194 (Fig. 5a). Translation of this protein was better with the uncoupled than with the coupled system (see also Fig. Sa). E l was originally identified in minicell studies (21). It has been shown to be a site-specific re~ombinase.~ We have noted, based on minicell studies with plasmids of varying copy num-

M. Gennaro and R. P. Novick, submitted for publication.

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ber, that El may be a~toregulated.~ If this autoregulation is transcriptional, this may explain the increased synthesis of E l in the uncoupled system. The CfoB fragment of pE194 contains both the replication region of pE194 and ORF C. Translation of this fragment yields E4 (Fig. 5 4 . A unique SphI site is present in ORF C, and SphI-cleaved DNA yields no E4 in the translation system (Fig. 4). While a HpaII site is also found near the NH2 terminus of ORF C, HpaII-cut plasmid does show E4 translation (Fig. 4) in the coupled system. This may indicate the loss of an Escherichia coli RNA polymerase-specific recognition element upon cleavage with HpaII. pE194 cut with CfoI, PstI, and CluI shows no change in E2, E3, or E4 synthesis, as expected.

Several ermC mutants with deletions in the leader region that have already been well characterized were tested in the system. Fig. 6 presents such a study. Deletions 21 and B2 lack portions of the leader region and partially or fully expose the initiation region of the methylase message. These mutants, therefore, make detectable levels of methylase in the absence of erythromycin and are unaffected by erythromycin in vivo (17). The results shown in Fig. 6 confirm the in uiuo minicell studies reported earlier (21). Deletion 17 is a mutant with a 120-base pair in-frame deletion in the coding region. The resulting mutant protein is smaller than the methylase. Since the leader and the methylase initiation regions are intact, the mutant protein (E3’) should still be regulatable by erythromycin. Fig. 6C presents translation of deletion 17. The mutant proteins (E2’ and E3’) are visible just above and below E4, respectively.

The system was also checked with the naturally constitutive plasmid pIM13 carrying the ermC’ determinant. In this plasmid 107 bases between the two ribosomal binding sites are deleted, leading to high level constitutive synthesis in uiuo (22). With increasing amounts of erythromycin, no induction of methylase is observed (Fig. 7). As expected, with higher concentrations of erythromycin, the translation is somewhat inhibited.

Although the induction observed in both the coupled and uncoupled systems (Fig. 4) suggests that induction is at the post-transcriptional level, this was investigated in greater detail (Fig. 8). At a low concentration of RNA polymerase no detectable methylase synthesis was observed. E l and E4 appear to be preferentially transcribed. Methylase synthesis is

‘ A. G. Shivakumar and D. Dubnau, unpublished results.

I n Vitro Study of ermC Translational Attenuation 1759

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FIG. 6. Translation of ermC deletion mutants with high and low Mg2+. Lames 1 and 2 contained 15 mM M$+, and lanes 3 and 4 contained 22 mM M$+. Lanes 1 and 3, no erythromycin; lanes 2 and 4 contained 0.05 pg/ml erythromycin. Panels A, B, and C show translation products of 821, AB2, and A17, respectively. In panel C, lanes 3 and 4, E2' is seen above E4. In lunes 2 and 4, E3' is seen below E4. C, shows translation products of pE194 DNA with 0.05 pg/ ml erythromycin. M, $29 proteins.

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FIG. 7. Translation of pIM13 DNA. 6 pg of pIM13 was translated with varying amounts of erythromycin. Lanes 1-6 contained, respectively, 0,0.02, 0.04, 0.16, 0.4, and 0.8 pg/ml erythromycin. The arrow indicates the position of the errnC' methylase protein. B, blank without added template; S, pIM13 minicell standard.

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FIG. 8. Translational induction of methylase. Various amounts of pE194 DNA were transcribed with varying amounts of E. coli RNA polymerase and then translated with 15 mM Mg2+ and 50 p M rifampicin. Lanes 1-3 contained, respectively, 0, 0.05, and 0.4 pg/ ml erythromycin. Panels a-c contained 3 pg of DNA and 0.5,1.5, and 3 pg of RNA polymerase, respectively. Panels d and e contained 1.5 pg of RNA polymerase and 5 pg (d ) and 1 pg ( e ) of pE194 DNA. Panel f shows coupled translation of 5 pg of pE194 DNA in the presence of 15 mM M P . The reaction volume was 25 pl and contained 3 pl of S-30 extract. B, blank (+ rifampicin). S, pE194 minicell standard.

observed only at higher concentrations of RNA polymerase and is clearly induced by erythromycin. For comparison, the coupled system is shown in panelf. These results demonstrate that the erythromycin-induction of the ermC methylase occurs post-transcriptionally. Comparison of panels 8d and 8e reveals that the system is also dependent on template.

Of the MLS antibiotics, only erythromycin and oleandomycin can induce ermC methylase synthesis in uiuo (10). This was verified using the translation system. Fig. 9a shows the coupled translation of pE194 with erythromycin concentrations from 0 to 12.5 pg/ml. There is a gradual increase in the synthesis of methylase with increasing concentrations of erythromycin. Induction of methylase occurs with as little as 0.025 pg/ml and peaks at 0.125 pg/ml. Translation is almost completely inhibited at 12.5 pg/ml. There is no induction of methylase with clindamycin or tylosin. With these antibiotics, inhibition of translation occurs with only 0.25 pg/ml antibiotic and almost full inhibition at 2.5 pg/ml. With oleandomycin, induction of methylase occurs at relatively higher concentrations (compared to erythromycin), peaking at 2.5 pg/ml, with only partial inhibition of translation evident even a t 12.5 pg/ml.

We have noted based on minicell studies that a protein with the electrophoretic mobility of E2 is specified by a DNA sequence that also encodes the ermC methylase (23). We have suggested that E2 arises from a reading frame shift near the end of the leader peptide, occurring a t a run of 6 A residues (Lys-Lys) directly preceding the UAA stop codon (7). Read- through therefore occurs, and a fusion protein is synthesized with 39 or 40 amino acids attached to the NH, terminus of the methylase. Peptide mapping showed that the methylase and the fusion protein are indeed read in the same frame (23). Inspection of the DNA sequence reveals that a -1 frameshift would place a translating ribosome in the ermC methylase reading frame. The interrelationship between E2 and E3 can be seen in the presence of increasing concentrations of erythromycin during pE194 translation (Fig. 10, a, b, c ) . A gradual

1760 In Vitro Study of ermC Translational Attenuation

E2 - E3 -

FIG. 9. Induction specificity of macrolides. Translation was with 15 mM M$+, 5 pg of pE194 DNA, and 2 p1 of S-30. al-a8 contained, respectively, 0, 0.025, 0.05, 0.125, 0.25, 0.5, 2.5, and 12.5 pg/ml erythromycin. bl-b5 contained 0.025, 0.05, 0.25, 0.5, and 2.5 pg/ml clindamycin. cl-c8 contained 0.025, 0.05, 0.125, 0.25, 0.5, 1.0, 2.5, and 12.5 pg/ml oleandomycin. d l 4 contained 0.05, 0.125, 0.25, 0.5, and 2.5 pg/ml tylosin. B, blank. M, 629 proteins.

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FIG. 10. Translation of the UAA mutant with high and low Mg2+. a and b, translation of pE194 DNA. c, parent of UAA mutant plasmid. d and e, UAA mutant plasmid. a and d contained 15 mM M$+, and b, c, and e contained 22 mM M e in the translation mix. 1-5 contained, respectively, 0, 0.01, 0.05, 0.25, and 0.5 pg/ml erythromycin. B, blank. S, pE194 minicell standard. M, 629 proteins.

increase in methylase synthesis (due to induction) is observed with a concomitant decrease in the synthesis of E2. The same relationship can be seen in several of the other figures in this study. This is predicted by the translational attenuation model and by the frameshift model for E2 synthesis, since stalling a ribosome in the leader would both induce methylase synthesis and decrease E2 synthesis. We further observed that in uitro translation of E2 was sensitive to M$+ concentration; at higher Mg2+, synthesis of E2 was increased (Fig.

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FIG. 11. Translation of SD2 mutant and ermC' with low and high Mg2+. Panels a, b, and c show results for the SD2 mutant, ermC' and ermC, respectively. Lanes 1 and 3, no erythromycin. Lanes 2 and 4,0.05 pg/ml erythromycin. Lanes 1 and 2,15 mM M e . Lanes 3 and 4,22 mM M$+. M, 629 proteins. B, blank.

10, a uersus b). This presumably is due to an effect of Mg2+ on the efficiency of readthrough. Any mutant with defective SD1 or with a deletion in the leader region would be expected to produce no E2 and, conversely, any mutant with defective SD2 (without interrupting or shifting the E2 reading frame) will produce the frameshift protein and not the methylase. Deletions 21 and B2 are both lacking portions of the leader region, and no E2 is observed a t higher M e in these constitutive mutants (Fig. 6).

We have constructed an additonal mutant with an in-frame UAA codon introduced immediately following the start codon for the leader peptide (Fig. 1). The frameshift model predicts that E2 would not be made by this mutant. Fig. 10, d and e, confirms this prediction. With the mutant, no E2 or E3 is made a t either high or low M$+ concentrations, whereas E4 is still made. As expected from the translational attenuation model and from in vivo results (19), no induction of methylase occurs. The parent plasmid of the UAA mutant (Fig. 1Oc) behaves as does pE194 at a high M e concentration. The pIM13 methylase gene (ermC') is closely related to ermC. As mentioned earlier, most of the ermC' leader region is deleted, and SD1 and SD2 remain in tandem (22). As a result, methylase synthesis is constitutive, and no E2 is made a t high or low Mg2+ (Fig. l lb ) . An SD2 mutant, in which the sequence AGAGGG of SD2 has been changed to ACCCGG, has been constructed5 (Fig. 1). This change results in loss of ribosome interaction a t SD2 but presumably not a t SD1. As expected, only the frameshift protein is produced. The translation of this plasmid with low M e produces very little E2, and increasing the M$+ concentration results in elevated levels of E2 (Fig. lla). Further support for the frameshift model can

D. H. Bechhofer and D. Dubnau, submitted for publication.


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FIG. 12. Stability of ermC transcript during translation. Two successive translations were carried out in these experiments. The first translation was coupled, contained 15 mM M$+ and no [35S]methionine, and was in the presence (panel B) or absence (panel A ) of erythromycin. The products were phenokCHCl3 extracted and EtOH precipitated. The resulting sample containing RNA was used as template in the second translation (uncoupled) in the presence of [35S]methionine and rifampicin. Both the translations were in a 20-pl volume and contained 2 pl of S-30. In lanes 1 and 2, the first translation contained no pE194 DNA. In the second translation, 2.5 pg of pE194 DNA transcribed with 3 pg of RNA polymerase was used. Lanes I , 3, and 5 contained no erythromycin in the second translation. Lanes 2, 4, 6, and 13-15 contained 0.05 pg/ml erythromycin in the second translation. Lanes 5 and 6 contained 22 mM M e , and the rest of the lanes contained 15 mM M F in the second translation. Lanes 7- 12 contained, respectively, 0.005,0.025,0.05,0.25,0.5, and 2.5 pg/ml erythromycin in the second translation. Lanes 13-15 contained, respectively, %, 3/5, and %, of the RNA from the first translation. M , 429 proteins.

be obtained from the results in Fig. 6C. The in-frame deletion mutant 17 produces shortened E2 and E3 products (E2' and E3') as noted above. E2' is made in small amount in low M$+. In the presence of erythromycin, E2' synthesis is de- creased, and E3' synthesis is induced. In the presence of high M$+, E2' is synthesized in large amounts and induction of E3' is unaffected.

We have shown that the addition of erythromycin stabilizes the ermC message (13), and recently this effect has been investigated in detail? It was, therefore, important to determine if the induction effect is directly on translation or is a consequence of enhanced transcript stability. This was studied by retranslating already translated message. The first part of the experiment consisted of coupled translations of pE194 with or without erythromycin and without [35S]methionine. The translation mix was phenol:CHC13 extracted and ethanol precipitated. The resulting sample containing RNA was translated for a second time (in the presence of [35S]methionine and rifampicin) with or without erythromycin. If there were an erythromycin-dependent stabilization of the message re- sponsible for the observed induction effect, the first translation with no erythromycin would be expected to yield unstable message which then would yield low amounts of methylase in the second translation, in comparison to the sample where erythromycin is present in the first translation. Fig. 12 shows such an experiment. Panel A contained no erythromycin, and

panel B contained 25 pg/ml erythromycin in the first translation. The second translation of either transcript in the presence of [35S]methionine showed no significant difference in the synthesis of methylase or frameshift protein whether or not erythromycin was present in the first translation. Therefore, the presence or absence of erythromycin had no effect on the stability of the transcripts obtained during the first translation. Occasional high levels of methylase in panel B in the absence of erythromycin in the second translation (for instance, lane 3) is probably due to erythromycin carried over from the first translation. In any event, the in uitro stability of the ermC mRNA is unaffected by erythromycin. This conclusion was further supported by a hybridization experiment using an ermC-specific probe. The strength of the ermC signal noted on Northern blots was nearly the same following translation in the presence or absence of erythromycin? Finally, this conclusion was confirmed by including [w3'P]ATP in the coupled reaction mix and analyzing the resulting transcripts by electrophoresis. This experiment indicated that the stability of the ermC transcript was unaffected by the presence of erythromycin (not shown).

The translational attenuation model was further tested by measuring methylase production using various proportions of methylated and unmethylated ribosomes and various concen-

6C. S. Narayanan, D. H. Bechhofer, and D. Dubnau, data not shown.

1762 I n Vitro Study of ermC Translational Attenuation

trations of erythromycin. It is known that methylation of ribosomes by MLS methylase offers resistance to erythromycin (24). To confirm that ribosomes determine erythromycin sensitivity in our system, ribosomes and S-100 prepared from BD404 and BD886 were cross-mixed and translation of ermC tested with or without induction (Fig. 13). BD404 carries no ermC gene. BD886 has a single chromosomal copy of ermC (25), and extracts were prepared from this strain grown in the presence of erythromycin. These strains offer a source of unmethylated and methylated ribosomes with no contami- nating plasmid DNA. Induction by erythromycin occurs only with unmethylated ribosomes (Fig. 13, A and B) , although this induction is poor compared to that obtained with the unfractionated S-30 extracts. Unmethylated ribosomes are sensitive to erythromycin (lunes A4 and B4) with either unmethylated or methylated S-100, whereas methylated ribosomes are resistant to erythromycin with either S-100 extract.

Further support for the translational attenuation model was obtained by adding synthetic oligodeoxyribonucleotides complementary to various segments of the ermC transcript to the translation system (Fig. 1). An oligonucleotide complementary to bases 53-68 (fragment 2) should free SD2 and hence should induce methylase synthesis. Oligonucleotides complementary to bases 10-27 (fragment 1) or 131-147 (fragment 3) would restrict ribosome interaction with their binding sites and would result in inhibition of induction. Fig. 14 shows the results of such an experiment. The coupled translation extracts contained 0.25-, 0.75-, 1.5-, or 2.5-pg fragments with or without inducing concentrations of erythromycin. With increasing amounts of fragment 1 or 3 there is a gradual inhibition of frameshift protein synthesis in the absence of induction and of methylase in the presence of induction. The inhibition of frameshift protein is more marked with fragment 1 than with fragment 3. Probably inhibition occurs less readily when pairing is to a coding sequence than with pairing to SD1 (26). Alternatively, this difference in inhibition may occur because fragment 3 but not fragment 1 must compete with a complementary sequence. With fragment 2, in the absence of

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FIG. 13. Methylation specificity. The reactions (25.~1 volume) contained 1 p1 of unmethylated ribosomes ( A and B ) , or 1 p1 of methylated ribosomes (C and D) and 2 p1 of unmethylated S-100 ( A and D) , or 2 p1 of methylated S-100 ( B and C), 15 mM M$+, 5 pg of pE194 DNA. Lanes 1-4 contained 0, 0.02,0.2, and 1.5 pg/ml erythromycin. M, 629 protein.

erythromycin, there is an oligonucleotide concentration-dependent increase in methylase synthesis with a concomitant decrease in the frameshift protein. In the presence of both fragment and erythromycin, there is a constant high amount of methylase synthesized, together with low synthesis of frameshift protein. These experiments were also done using a high concentration of the fragments (Fig. 15, left). High levels of the fragments 1 and 3 generally inhibit translation (lunes 3 and 6). It is possible that at high concentrations these oligonucleotides bind indiscriminately to Shine-Dalgarno sequences. Finally, these effects were studied using the uncoupled translation system (Fig. 15, right). pE194 transcripts were preincubated with the fragments and then translated in the presence of rifampicin. Oligonucleotide 2 shows an induction effect in the uncoupled system. The inhibitory effect of fragments 1 and 3 is more pronounced than in the coupled system, possibly because in the latter ribosomes can compete more effectively with the oligonucleotide fragments for binding to nascent mRNA.

DISCUSSION

In order to study the translational attenuation model in greater detail, we have used an in vitro translation system from B. subtilis. In this system the proteins specified by individual ORFs can be verified by the use of DNA fragments containing a given ORF or appropriate linearized plasmids. In general, the i n vitro system faithfully imitates in vivo behavior. However, there are some exceptions. For example, protein E4 from the rep region of pE194 is not detectable in minicells. In in vitro translation it is the most prominent protein, suggesting that an important regulation is completely lost in vitro. Another drawback in this system is the apparent failure to detect truncated proteins. Plasmids cut with HpuI, BclI, and Hue111 should give rise to truncated methylase products, but these were not observed. On the other hand, we have found that if a circular template carrying cloned fragments is used, we can detect truncated proteins (not shown).

Previous experiments have demonstrated the synthesis of E2 in vivo and have suggested that it is a fusion protein resulting from a translational frameshift (7,23). In the present study we have shown that E2 is also synthesized in vitro. E2 is not produced by suppressor activity in the E. coli tRNA, permitting readthrough at the leader peptide stop codon, since it is produced in the absence of added tRNA (not shown). The significance of E2 i n vivo is not known. E2 synthesis i n vitro can be increased by raising the Mg2+ concentration. It is possible that high Mg2+ increases the frequency of reading frame shifting during translation of the leader peptide. We believe that this shift may occur at the run of 6 As immediately preceding the leader peptide stop codon. If so, the shift must be context dependent, since ermC contains a stretch of 7 As a t position 870, and a frameshift at that position would introduce stop codons 20-30 bases downstream, yielding a fusion protein of 27 or 28 kDa (50-60 amino acids less than E2). No such product has been observed. The Mg2+-dependent increase in the synthesis of E2 had no effect on the induction of methylase, since equal amounts of the latter are made at high or low M F . On the other hand, the increase in methylase during induction is accompanied by a gradual decrease in frameshift protein synthesis. A simple explanation based on the translational attenuation model is that erythromycin- bound ribosomes stall during induction and prevent translation of E2 (see accompanying paper (38)).

Our laboratory has presented in vivo evidence showing that induction of methylase is a post-transcriptional regulatory event (13). In this paper we have confirmed this by using the

In V i t ro S tudy of ermC Translational Attenuation

FIG. 14. Effect of synthetic oligonucleotide fragments on methylase synthesis. The reactions contained 2 pg of pE194 DNA, 15 mM M F , the various synthetic oligonucleotide fragments, and 2 pl of S-30. Incubation was at 37 'C for 30 min. One-fourth of each reaction was electrophoresed. Lunes 1-4 contained no erythromycin, and lanes 5-8 contained 0.05 pg/ml erythromycin. The amounts of fragments used are: lanes 1 and 5,0.25 pg; lanes 2 and 6,0.75 pg; lanes 3 and 7, 1.5 pg, and lanes 4 and 8, 2.5 pg. - and + refer to control translations of pE194 DNA containing no oligonucleotide and with, respectively, no erythromycin and 0.05 pg/ml erythromycin. M,, molecular weights of 429 proteins. B, blank.

E2 E;!

FI F2

FI F 2 "

R - + 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 0

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F3 - 1 2 3 4 5 6 7 8 Mr

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FIG. 15. Effect of synthetic oligonucleotide fragments at high concentrations on methylase synthesis. Coupled translation (left): the reactions contained 2 pg of pE194 DNA, 15 mM M$+, and varying amounts of synthetic fragments. The reactions were started by adding 2 pl of S-30. The incubations were at 37 "C for 30 min. Uncoupled translation (right): RNA from transcriptions with 2 pg of pE194 DNA and 3 pg of E. coli RNA polymerase was mixed with varying amounts of fragments. The general components, rifampicin and 15 mM Mg2' were added, and the mixture was annealed by incubation at 37 "C for 10 min, 42 "C for 2 min, and cooling to 4 "C. This RNA was translated with 2 pl of S-30 at 25 "C for 60 min. One-fourth of each reaction mix was electrophoresed. Lanes 1-4, no erythromycin; lanes 4-6, 0.05 pg/ml erythromycin. 1, and 4, 2.5-pg; 2, and 5, 7.5-pg; and 3, and 6, 12-pg fragments used. - and + show translation of 2 pg of pE194 DNA with no oligonucleotide and without or with 0.05 pg/ml erythromycin, respectively. M, 429 proteins.

uncoupled translation system. The inducing concentration of erythromycin i n vitro (0.02-0.05 pg/ml) is similar to that used i n vivo. The in vitro system is also accurate with regard to inducer specificity. Only erythromycin and oleandomycin can induce methylase. With oleandomycin, the optimal concentration for induction seems to be high compared to erythromycin. A similar observation has been reported i n vivo (27). Although induction of methylase has been shown to be post-

transcriptional, it need not necessarily occur by the direct stimulation of translation, since the erythromycin-dependent stabilization of ermC transcript in vivo has been established (13). It would be difficult to show that induction occurs by the stimulation of translation using in vivo experiments. However, we have now demonstrated i n vitro that erythromycin has little effect on the stability of the ermC transcript. In fact, it appears that i n vitro the ermC mRNA is more stable

1764 In Vitro Study of ermC Translational Attenuation

than in vivo in the absence of erythromycin. It is possible that during S-30 preparation, crucial RNases cosedimented with the cell debris or that the concentrations of important components of the degradative machinery are below K,,, levels in vitro. Our extracts are not completely devoid of RNase activity, and this has been exploited in the accompanying paper (38) to study the stalling of ribosomes on the ermC transcript. Upon longer incubation of the RNA with the translating extract (greater than 60 min), RNA instability can be demonstrated in vitro, but this is not specific to ermC RNA and does not appear to be affected by erythromycin. Therefore, it is concluded that the induction of methylase by erythromycin in vitro is exerted translationally. However, in vivo both mRNA stability and translational induction may contribute to the erythromycin effect on methylase synthesis.

According to the translational attenuation model, if a ribosome is stalled while translating the leader peptide, induction takes place. Stalling, therefore, requires an unmethylated erythromycin-bound ribosome. However, an uninhibited ribosome, methylated or unmethylated, must be available to translate from SD2. These requirements have been explored by mixing unmethylated and methylated ribosomes in various proportions at various erythromycin concentrations. The probability of stalling an erythromycin-bound ribosome is increased at high erythromycin concentration and with higher proportions of unmethylated ribosomes. These conditions, on the other hand, also favor inhibition at SD2. An optimum should, therefore, be observed at some intermediate proportion of unmethylated and methylated ribosomes with a given erythromycin concentration. This effect can be clearly seen in Fig. 1 in the Miniprint. Particularly dramatic is the effect of adding even a small proportion of methylated extract. This permits a marked increase of induction at moderate to high concentrations of erythromycin. This is to be expected from the considerations mentioned above. These effects are specific for the inducible ermC and are not seen with the constitutive ermC'.

We have used synthetic oligonucleotides as probes in testing the translational attenuation model. Any event that frees SD2 from base pairing should lead to induction. This can be accomplished by ribosome stalling or by the use of oligonucleotide 2 (Figs. 1, 14, and 15). Oligonucleotides 1 and 3 inhibit induction, confirming the role of SD1 and SD2 in the induction process.

We have shown in this study that the in vitro system is a powerful tool for analyzing ermC regulation. We have demonstrated that the erythromycin-dependent induction of methylase is due to increased translation that occurs in the absence of any effect on message stability. The unmethylated and methylated ribosome mixing experiments and studies with complementary oligonucleotide probes provide strong and direct support for the translational attenuation model.

Acknowledgments-We thank J. S. Krakow (Hunter College, NY) for a generous gift of E. coli RNA polymerase and L. Mindich for advice in setting up the S-30 system and for valuable discussion. We thank A. Howard for expert secretarial assistance.

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E X P E R I M E L L PROCEDURES RESULTS AND DISCUSSION

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An in Vitro Study of the Translational Attenuation Model of ermC