From the Department of Chemistry, Moscow State University, 119899 Moscow, Russia and the ¶Max Planck Institut fur Molekulare Genetik, 14195 Berlin, Germany
Received for publication, March 20, 2003 , and in revised form, April 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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Two methods have been published describing attempts to create a pure population of ribosomes carrying lethal mutations (710). Both of them involve in vitro transcription of an rDNA or rDNA fragment and subsequent reconstitution of the ribosomal subunit. However, the first method (9) uses ribosomes that are not from E. coli, and the second (7) utilizes fragmented rRNA, leading to very low activity (for discussion, see Ref. 10). The major problem here lies in the absence of natural modifications as well as possible (at least partial) misfolding of the rRNA in vitro, as compared with the in vivo assembly.
We aimed to create a system for affinity purification of ribosomes, assembled in vivo, via an aptamer tag attached to the mutated rRNA. Whereas the purification of proteins carrying genetically introduced affinity tags has become a routine, only a few such tags are known for RNA (1113), and none of them has so far been applied to a ribonucleoprotein particle with the size of the ribosome.
One of the most challenging steps of the protein synthetic cycle is translocation, and the coupling of GTP hydrolysis and translocation, stimulated by elongation factor G, has been widely discussed in the literature. There are two known sites of interaction of EF-G1 with the 23 S rRNA, namely the sarcinricin loop (SRL) and the GTPase-associated center (14). The mutation G2655C of the SRL of 23 S rRNA is known from the literature to be lethal (15), an effect that is related to the interaction of the elongation factor with the ribosome. In this report, we decided to study this mutation in more detail, namely to find an exact stage in the elongation factor G cycle affected by the G2655C mutation. For this purpose, we applied a method for the affinity purification of ribosomes harboring the G2655C lethal mutation in an amount and purity sufficient for biochemical characterization.
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EXPERIMENTAL PROCEDURES |
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Mutations of nucleotide 2655 of the 23 S rRNA were made by site-directed mutagenesis of an SphI-BamHI fragment of the E. coli rrnB operon, subcloned into M13 mp18 phage. Subsequently, the mutated fragment was subcloned back into the pLK1192U plasmid either with or without the affinity tag.
The XL1 strain of E. coli was used for all genetic manipulations and phage growth. For preparation of uracil-containing single-stranded DNA for mutagenesis, strain CJ236 was used as a host for M13 phage. For translation fidelity measurements, strain MC140 was used. Strain AVS69009 (4) carrying no chromosome-encoded rRNA was transformed by pLK1192U plasmids carrying mutations. For displacement of originally present pHK-rrnC plasmid, the transformants were streaked on LB plates containing spectinomycin (pLK1192U encodes a mutation in 16 S rRNA, causing resistance to spectinomycin). The loss of the pHK-rrnC plasmid was monitored by loss of kanamycin resistance. The absence of wild type rRNA in the cell was confirmed by the primer extension method, as in Sergiev et al. (5). The primer was complementary to nucleotides 548564 of the 23 S rRNA. The extension was carried out in the presence of dATP, dCTP, dTTP, and ddGTP, thus proceeding up to nucleotide C544 in the case of the wild type and until the last cytosine of the insert, 2 nucleotides from the primer binding site in the case of ribosomes carrying the affinity tag.
Translational Fidelity Measurements and Growth Rate
DeterminationCells from strain MC140, transformed both by
pLK1192U, carrying a mutation at nucleotide 2655 of the 23 S rRNA, and by the
pSG or CSH series of plasmids
(19), were grown at 37 °C
until an optical density at 600 nm of 0.3 was reached. -Galactosidase
activity was assayed essentially according to O'Connor et al.
(20).
Growth rates were measured at 37 °C in LB media containing 50 µg/ml ampicillin. Three independent colonies were grown overnight after transformation of the XL1 strain or plasmid substitution in the AVS69009 strain and were used to inoculate a volume of medium 50 times larger. Cell densities were monitored by absorbance at 600 nm.
Ribosome Preparation and Affinity PurificationRibosomes were prepared by the reassociation method (21). Affinity purification was carried out in subunit dissociation buffer (20 mM Hepes-K, pH 7.5, 1 mM Mg(OAc)2, 200 mM NH4C1, 4 mM 2-mercaptothanol) at 4 °C. Binding of 1000 pmol of ribosomes to 100 µl of streptavidin-Sepharose (Amersham Biosciences) was performed overnight (16 h) in a 2-ml tube followed by three washes with binding buffer. Elution was also carried out overnight, using binding buffer saturated with biotin. After elution, the 50 S ribosomal subunits were reassociated with 30 S subunits from an additional subunit preparation, and the 70 S ribosomes were separated by sucrose density gradient centrifugation. The ratio of wild type to mutant in the ribosome preparations was estimated by primer extension, according to Sigmund et al. (22).
GTPase ReactionFor the GTPase reaction, 4 pmol of ribosomes
were mixed with a preincubated (10 min, 37 °C) mixture of 4 pmol of EF-G,
4 pmol of GTP, and 0.1 µCi of [-32P]GTP for one round of
hydrolysis and 400 pmol of GTP for multiple rounds of hydrolysis. Buffer
conditions for the reaction were 20 mM Hepes-K, pH 7.5, 10
mM MgCl2, 100 mM NH4Cl, 4
mM 2-mercaptoethanol. Fusidic acid was added if required to a final
concentration of 1 mM. 40% formic acid was used to stop the
reaction. The extent of GTP hydrolysis was assayed by thin-layer
chromatography (23).
Recombinant EF-G was purified from the superproducing strain
(24), provided by Dr. T.
Ueda.
FootprintingFor footprinting experiments, the complexes of ribosomes and EF-G with or without fusidic acid were prepared as for the GTPase reaction (above) but without radioactive GTP. Dimethylsulfate (DMS) modification was carried out by addition of 0.2 µl of 20% DMS solution in ethanol to 10 µl of the complex. Modification was allowed to proceed for 10 min at 37 °C. The reaction was stopped, rRNA was extracted, and primer extension was carried out as described in Sergiev et al. (5). For the analysis of the region around the SRL and GTPase-associated center, primers complementary to 23 S rRNA nucleotides 11211140 and 27302749, respectively, were used.
Translocation ExperimentsFor translocation experiments, 2.5 pmol of ribosomes were mixed with 1 µg of polyU (Sigma) and a 2-fold excess of tRNAPhe in a 10-µl volume of polyamine buffer (20 mM Hepes-K, pH 7.5, 6 mM Mg(OAc)2, 150 mM NH4OAc, 2 mM spermidine, 0.05 mM spermine, 4 mM 2-mercaptothanol) (21) and incubated at 37 °C 15 min. After incubation, a 1.2-fold excess of AcPhe-tRNAPhe in the same buffer was added followed by incubation for an additional 15 min. The same buffer with or without equimolar amounts of EF-G and GTP (2 mM final concentration) was added, and incubation was continued for a further 10 min at 37 °C. In parallel, the binding of AcPhe-tRNAPhe directly to the P-site was done the same way but without prior incubation of the ribosomes with the deacylated tRNA. Binding of AcPhe-tRNAPhe to the ribosomal P or A sites was measured by filtration through nitrocellulose. The puromycin reaction was performed at 37 °C for 10 min with a 5 mM final concentration of puromycin adjusted to pH 7.5. The radioactive amino acid released was extracted with ethyl acetate. The background of the puromycin reaction was measured in a parallel experiment without puromycin.
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RESULTS |
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Introduction of the MS2 coat protein binding site into rRNA has been described previously. A recombinant fusion protein consisting of the MS2 coat protein dimer (11), a TEV-protease cleavage site, and a dimer of the Z domains of Staphylococcus aureus protein A was created for the affinity purification. Using this system, we were able to observe a quantitative binding of the fusion protein to the affinity-tagged ribosomes and, independently, to the IgG-Sepharose (data not shown). However, the binding of ribosomes carrying the MS2 coat protein binding site to the resin, mediated by the recombinant protein, was extremely low and relatively non-specific (data not shown). The reason was most probably in the steric clash between ribosomes, protein, and the resin when all three are to bind each other simultaneously. The successfully applied MS2 coat protein-based systems for affinity separation of RNA and its complexes obviously involved ribonucleoprotein particles of a much smaller size. We also cannot rule out that more flexible and long linkers between the MS2 coat protein part and the IgG-Sepharose binding part of the fusion protein could potentially enable the binding of tagged ribosomes to the resin.
The next affinity tag to be tested was the streptomycin binding aptamer (12), which was introduced into helix 98 of the 23 S rRNA. Purification of the ribosomes carrying this aptamer on immobilized dihidrostreptomycin gave a very high level of co-purification of wild type ribosomes (data not shown). This might be explained by binding of the streptomycin derivative to the 30 S ribosomal subunit or by non-specific electrostatic interactions between the dihydrostreptomycin and rRNA. The latter is more probable since the application of the 50 S subunits instead of the 70 S ribosomes for the binding does not increase the specificity of binding.
Finally, an aptamer to streptavidin (13) was introduced into the helices 9, 25, and 45 of the 23 S rRNA. Strains (4) expressing only the tagged variant of 23 S rRNA had no growth defects in rich medium, indicating that tagged ribosomes could support translation equally to the wild type ribosomes. Pure tagged ribosomes bound streptavidin-Sepharose and could be eluted by biotin in a similar manner as that described for yeast RNase P (13). The best binding was observed when the aptamer was introduced into helix 25 (Fig. 1). In equal conditions, binding of other variants of tagged ribosomes was 70% of the binding of the ribosomes carrying the aptamer inserted into the helix 25. Under equal conditions, the ribosomes without the introduced aptamer were not able to bind the resin to any detectable extent.
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Affinity Separation of Ribosomes Carrying the Lethal G2655C MutationPlasmid pLK1192U, encoding 23 S rRNA carrying both the streptavidin aptamer and the G2655C mutation, was expressed in strain XL1 of E. coli. The plasmid used carries the ampicillin resistance gene and C1192U mutation in 16 S rRNA, making ribosomes resistant to spectinomycin. 70 S ribosomes from this strain were prepared by the reassociation technique (21). Affinity purification of tagged ribosomes by streptavidin-Sepharose at 10 mM magnesium concentration lead to only 5-fold enrichment of the initial mixture. Since binding of pure wild type ribosomes (not as a mixture with tagged ones) to the resin was not observed, we concluded that non-specific binding of non-tagged species arises from ribosome-ribosome rather then ribosome-resin interactions. To avoid this effect, we purified 50 S subunits at a magnesium concentration of 1 mM, which led to a satisfactory separation; the initial mixture contained only 6% tagged ribosomes, which was enriched to a level of 90 ± 3% in the final eluate from the resin (Fig. 1). The affinity chromatography step was followed by reassociation of the subunits and further purification of the 70 S ribosomes by ultracentrifugation in a sucrose density gradient as described by Blaha et al. (21).
The G2655C Mutation Affects Cell Growth and Accuracy of TranslationIn agreement with previous reports (15), we found that G2655C mutation is recessive lethal. It was not able to support cell growth if no wild type ribosomes were present in the cell; however, it could be tolerated if the translation is carried out by wild type ribosomes. In a strict sense, the mutation did not allow AVS69009 strain (4) to lose the plasmid encoding the wild type 23 S rRNA and remain viable. The study was extended to other substitutions of the 2655 nucleotide, namely G2655A and G2655U. In contrast to G2655C, these mutations were found to be viable and were able to support growth, even when they represented 100% of the 23 S rRNA in AVS69009 cells in which all the chromosomal rDNA operons are deleted. The doubling times of the corresponding strains are listed in Table I. In agreement with previous reports (25), the G2655A mutation showed little difference from the wild type, G2655C was the most defective, and G2655U was intermediate.
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The effect of the mutations at 23 S rRNA position 2655 on the in
vivo accuracy of translation was tested with the help of a set of
plasmids expressing the -galactosidase gene
(19). Each plasmid of this set
(except pSG25, used as a control) contain a mutation in the lacZ gene
that would lead to a non-functional protein if the translation of
correspondent mRNA proceeded without mistakes. The active enzyme could only be
synthesized as a result of a translation error, such as frameshifting, stop
codon readthrough, and misreading, compensating the mutation encoded in the
DNA. Stop codon readthrough and frameshifting was increased in the strains
expressing mutant rDNA (Table
II). The extent of the increase correlated well with the growth
rates, being minimal for G2655A and maximal for the lethal G2655C mutation.
Curiously, the mutations also increased the expression of the normal
-galactosidase gene, an effect that has been described in the literature
for several other mutations and explained by an increase of overall wild type
ribosome production in response to the expression of mutant rDNA genes
(26).
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The interesting observation is the decrease in Glu/Gln misreading in strains expressing the G2655C and G2655U mutants (Table II). Altogether these effects could be explained if we suppose that the G2655C mutant ribosomes move very slowly along the mRNAs. In this case, the downstream ribosomes in the polysomes would be forced to move as slowly as the mutant ones, thus gaining additional time for correct tRNA selection as well as for shifting the reading frame.
Ribosomes Carrying the G2655C Mutation Stimulate GTPase of EF-GThe experiments were carried out with ribosomes, purified by affinity chromatography, carrying the G2655C mutation. Ribosomes, purified by the same technique, with a wild type 2655G nucleotide were used as a control.
The multiple EF-G turnover reaction was measured at a ribosome:EF-G:GTP ratio of 1:1:100. It was shown that ribosomes carrying the G2655C mutation have a slightly slower GTPase turnover rate (Fig. 2). However, in contrast to our expectations, it was found that the mutant ribosomes do in fact stimulate the GTPase activity of elongation factor G. According to the experiments of Munishkin and Wool (25), a sarcin-ricin loop oligonucleotide carrying the G2655C mutation does not bind EF-G. We decided to check whether the same is true in the context of the ribosome. It is known that fusidic acid stabilizes the complex of EF-G with the ribosome, where EF-G forms a contact with the SRL (14, 27). We checked the inhibition by fusidic acid of GTPase turnover on the mutant ribosomes and found it to be the same as for the wild type ribosomes. Since fusidic acid prevents dissociation of EF-G and the ribosome, this inhibition proves that EF-G binds to the ribosome. This might indicate that an interaction of EF-G with other parts of the ribosome, most probably with the GTPase-associated center, could overcome the effect of the mutation and be sufficient for EF-G binding.
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EF-G Contacts GTPase-associated Center of Ribosomes Carrying the G2655C Mutation, whereas the Interaction with the Sarcin-Ricin Loop Is ReducedStimulation of the GTPase activity already presumes that EF-G binds to the mutant ribosomes. However, to demonstrate EF-G binding directly and to see whether it contacts both the SRL and the GTPase-associated center, we checked the protection by EF-G of 23 S rRNA residues from DMS modification, with and without fusidic acid.
We found that ribosomes carrying the G2655C mutation could bind EF-G (Fig. 3), although the extent of binding is reduced in comparison with the binding to wild type ribosomes. We conclude that EF-G contacts both the SRL and the GTPase-associated center of the mutant ribosomes since both A1067 (in the GTPase-associated center) and A2660 (in the SRL) were protected from DMS modification by EF-G. Notably, protection of A1067 in both the wild type and mutant ribosomes is almost the same, whereas the extent of A2660 protection varied somewhat from one experiment to another, being on average 80% for the wild type and 31% for the mutant in the case of the complex with the fusidic acid. Without the antibiotic, EF-G-related protections on both the wild type and mutant ribosomes were reduced in agreement with the literature data (14). It was not possible to compare the reactivity of nucleotide 2655 in mutant versus wild type ribosomes since only the mutant is reactive to DMS. However, it is noteworthy that, whereas G2655 of wild type ribosomes is protected by EF-G from modification with kethoxal (14), C2655 of the mutant ribosomes is not protected from DMS modification.
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Ribosomes Carrying the G2655C Mutation Are Defective in EF-G-driven TranslocationThe most intriguing question to ask was whether EF-G could cause the translocation of tRNAs on the mutant ribosomes. To test this, we prepared a pretranslocation complex on mutant polyU-programmed ribosomes with the P-site filled by deacylated tRNAPhe and the A-site filled by AcPhe-tRNAPhe. In parallel, AcPhe-tRNAPhe was directly mixed with polyU-programmed ribosomes so as to observe its maximal level of binding to the P-site without prior incubation with deacylated tRNAPhe. The proportion of P-site-bound AcPhe-tRNAPhe was measured by the puromycin reactivity of the AcPhe moiety related to the puromycin reactivity of AcPhe-tRNAPhe bound directly to the P-site. The experiments were done according to the modified Watanabe system (21, 28) in polyamine-containing buffer. As expected, no detectable difference between G2655C and wild type ribosomes with regard to P- and A-site tRNA binding was observed. Binding of AcPhe-tRNAPhe to the P-site of polyU-programmed ribosomes was on average 98%, whereas the binding to the A-site of ribosomes carrying deacylated tRNAPhe in the P-site was 50%, both values being identical for wild type and G2655C ribosomes. Translocation, both spontaneous and EF-G-related, was measured by the puromycin reaction (Fig. 4) with and without addition of EF-G. No difference in spontaneous translocation was detected, but a clear defect (circa 3-fold difference) in EF-G-driven translocation was observed for the G2655C ribosomes. Under the same incubation conditions, a 100-fold excess of GTP is completely hydrolyzed by EF-G, stimulated by ribosomes carrying the G2655C mutation. Thus, the principal result is that the G2655C mutation moderately affects GTPase stimulation and binding of EF-G to the ribosome but significantly decreases translocation efficiency.
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DISCUSSION |
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Streptomycin binding aptamer was introduced to the helix 98 of the 23 S rRNA. It was shown not to be suitable for the affinity preparation of mutant ribosomes due to the high background binding of wild type ribosomes to the immobilized dihidrostreptomycin. Such a low specificity might be explained by electrostatic binding of large ribosomal RNA to the positively charged streptomycin.
An aptamer to streptavidin inserted to the helix 25 of the 23 S rRNA performed the best as an anchor to the mutant ribosomes. It was demonstrated that the only limitation to the purity of the mutant ribosomes separated by this technique of affinity chromatography resides in ribosome-ribosome interactions. Application of the low magnesium buffer, reducing unspecific interactions between ribonucleoprotein particles, allowed us to separate tagged 50 S ribosomal subunits sufficiently pure for the biochemical analysis in vitro.
The G2655C mutation in 23 S rRNA was selected to test the method. Nucleotide G2655 is protected by both EF-G and EF-Tu from chemical modification (14). This nucleotide is not involved in extensive intraribosomal interactions (1), so its mutation would not be expected to cause any conformational changes outside the elongation factor binding site. The mutation G2655C was first described for the SRL oligonucleotide (25) and was shown to impede EF-G binding to this RNA fragment. Later, Macbeth and Wool (15) showed that expression of 23 S rRNA carrying this mutation, from highly efficient P1P2 rDNA promoters, causes cell lethality. The mutant ribosomes were shown to be no more than 3% active in protein synthesis (15). Mutations of other SRL nucleotides have defects in EF-G binding (29) and EF-Tu/AatRNA/GTP ternary complex binding (30), as well as effects on translation fidelity (31, 32). The mutations described here also affect the fidelity of translation, and they do so in opposite directions; stop codon readthrough and frameshifting are increased, whereas misreading is decreased. Both effects could be explained if one postulates a slower movement of the mutant ribosomes along an mRNA molecule. An overall decrease in translation speed would result in a wider time window for correct tRNA choice, as well as for frameshifting.
Despite the large body of experimental data, the mystery of the function of the elongation factors on the ribosome is still unsolved. One of the most intriguing questions is the coupling of GTP hydrolysis with translocation. In the classical view (33, 34), translocation takes place before GTP hydrolysis, the latter being only necessary to dissociate the factor. A more recent, but often disputed, view supports GTPase activation prior to translocation (35). Additional support for this alternative hypothesis has come from biochemical (36) and cryo-EM (37) studies but has, however, been criticized by Cameron et al. (38).
Here, we present a unique analysis of lethal mutant (G2655C) ribosomes purified by affinity chromatography, showing GTPase activity and EF-G binding accompanied by significantly inhibited translocation. The binding of EF-G to the GTPase-associated center was the same for wild type and the G2655C ribosomes; however, the contact of EF-G with SRL of the mutant ribosomes was weaker. This result could be explained in several ways. In the context of the model of Rodnina et al. (35), our results indicate that EF-G is in contact with nucleotide 2655 after the GTPase reaction but prior to translocation, as shown by the cryo-EM study (37). Thus, translocation is not necessary for GTPase activation, simply because we observe GTPase activity without translocation. However, the results obtained could also be explained in terms of the classical model, which supposed a mechanism whereby GTP hydrolysis is activated only after the translocation has taken place. Thus, it could be hypothesized that the mutation in residue 2655 allows the necessity for translocation to activate the GTPase activity to be "bypassed." In other words, the mutation could make GTPase and translocation uncoupled, in the way that it occurs with EF-G with the deleted domain IV (39). It is also possible that the G2655C mutation could impede an allosteric signal transition from the sarcin-ricin loop to the peptidyltransferase center, thus making it impossible for the CCA ends of the tRNA molecules to shift. Both explanations converge at the point that interaction between EF-G and SRL is not primarily essential for the activation of GTP hydrolysis; however, it is important for translocation. The method described for affinity chromatography of ribosomes will undoubtedly help to explore more lethal mutations, giving insight into other critical steps in the ribosomal function.
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FOOTNOTES |
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Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.: 7-095-9395418; Fax:
7-095-9393181; E-mail:
petya{at}genebee.msu.su.
1 The abbreviations used are: EF, elongation factor; SRL, sarcin-ricin loop;
DMS, dimethylsulfate; TEV, tobacco etch virus; WT, wild type.
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ACKNOWLEDGMENTS |
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REFERENCES |
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