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Translation of leaderless mRNAs, lacking ribosomal recruitment signals other than the 5′-terminal AUG-initiating codon, occurs in all three domains of life. Contemporary leaderless mRNAs may therefore be viewed as molecular fossils resembling ancestral mRNAs. Here, we analyzed the phenomenon of sustained translation of a leaderless mRNA in the presence of the antibiotic kasugamycin. Unexpected from the known in vitro effects of the drug, kasugamycin induced the formation of stable ~61S ribosomes in vivo, which were proficient in selectively translating leaderless mRNA. 61S particles are devoid of more than six proteins of the small subunit including the functionally important proteins S1 and S12. The lack of these proteins could be reconciled with structural changes in the 16S rRNA. These studies provide in vivo evidence for the functionality of ribosomes devoid of multiple proteins, and shed light on the evolutionary history of ribosomes.
The rate limiting step and major checkpoint of translational regulation in Bacteria is the formation of the translation initiation complex, comprising the 30S ribosomal subunit, and mRNA (Gualerzi and Pon, 1990). The efficiency of formation of this ternary complex is modulated by intrinsic features of the 5′-untranslated region (UTR) of the mRNA, including the Shine and Dalgarno (SD) sequence and a region rich in pyrimidines, which is recognized by ribosomal protein (r-protein) S1 (reviewed in Laursen et al., 2005). In contrast, leaderless mRNAs (lmRNAs), which are present in Bacteria, Archaea and Eukarya, start directly with the AUG-initiation codon, and thus lack these recognition motifs. As there is no evidence for ribosome recruitment signals in the 5′-coding region of lmRNAs (reviewed in Moll et al., 2002b), the 5′-terminus with its start codon represents the only constant recognition element for the ribosome.
Translation of lmRNAs was stimulated by translation initiation factor 2 (IF2; Grill et al. 2000, 2001), which facilitates binding to the ribosome. These data were interpreted as showing that the 5′-terminal start codon of lmRNAs is recognized by bound to ribosomes. In addition, IF2 stimulates 30S-50S subunit association (La Teana et al., 2001; Antoun et al., 2003), which offered an additional explanation for the stimulating effect of IF2 via the 70S initiation pathway (Moll et al., 2004). A number of observations support the view that 70S ribosomes initiate translation of lmRNA: (i) Recent experiments revealed that intact 70S monosomes are competent for translation initiation of lmRNAs (Moll et al., 2004; Udagawa et al., 2004), whereas (ii) overexpression of IF3 triggering dissociation of the ribosomal subunits was shown to exert a negative effect on lmRNA translation (Moll et al., 2004; Grill et al., 2001). (iii) The 70S ribosome translation initiation pathway of lmRNAs was supported by a set of experiments using ribosomes devoid of the r-proteins S1 and S2. These S1/S2 deficient monosomes were shown to be particularly stable and to perform selective translation of lmRNA (Moll et al., 2004).
The aminoglycoside antibiotic kasugamycin (Ksg) inhibits translation at the step of initiation complex formation in Pro- and Eukaryotes (Okuyama et al., 1971). Recently, the binding sites of Ksg on Thermus thermophilus 30S ribosomal subunit as well as on the E. coli 70S ribosome were identified by X-ray crystallography (Schluenzen et al., 2006, Schuwirth et al., 2006). These studies located a primary Ksg binding site on top of helix 44 (h44), spanning the region between h24 and h28 of 16S rRNA, which contacts the conserved nucleotides A794 and G926. This position coincides with the mRNA track at the P- and E-site codon. Binding experiments indicated that Ksg indirectly induces dissociation of P-site bound from 30S subunits through perturbation of the mRNA, thereby interfering with translation initiation (Schluenzen et al., 2006, Schuwirth et al., 2006).
In contrast to the inhibitory effect of Ksg on translation of canonical mRNAs, translation of lmRNAs appeared to prevail in the presence of the drug in vivo (Chin et al., 1993; Moll and Bläsi, 2002). In this study, we have scrutinized the molecular mechanism(s) underlying translation of lmRNA in the presence of Ksg. Our data revealed that during lmRNA expression Ksg induces in vivo the formation of ribosomal particles lacking several proteins of the small subunit, hereafter termed “61S particles”. We demonstrate that these protein-deficient ribosomes selectively translate lmRNAs in the presence of the drug in vivo and in vitro, which provides evidence for the in vivo functionality of ribosomes lacking several proteins. In addition, chemical probing studies are presented, which suggest a model for the formation of these protein-deficient particles in the presence of the drug.
We and others (Chin et al., 1993; Moll and Bläsi, 2002) observed that the activity of λ CIΦLacZ fusion proteins encoded by lmRNAs continued to increase in the presence of Ksg. To verify the apparent resistance of lmRNA translation to Ksg, we first performed a pulse labeling experiment in the presence of 750 μg/ml Ksg using E. coli strain MG1655 harboring plasmid pRB381-1, which encodes a leaderless cI-lacZ fusion gene. This concentration of Ksg drastically reduced growth (data not shown). As shown in Figure 1A, lanes 5-7, translation of bulk mRNA was strongly diminished upon addition of Ksg, whereas in striking contrast, the de novo synthesis of the CIΦLacZ fusion protein continued in the presence of the drug. However, we were unable to recapitulate these results in vitro by programming an E. coli in vitro translation system with both, the leaderless λ cI mRNA and E. coli ompA mRNA, the latter of which contains a 135 nt long 5′-UTR, containing a canonical ribosome binding site (Rosenbaum et al., 1993). In contrast to the in vivo experiment (Figure 1A), increasing concentrations of Ksg inhibited in vitro translation of both mRNAs (Figure 1B, lanes 2-5).
70S initiation complexes on lmRNA rather than 30S ternary complexes were shown to be comparatively resistant to Ksg (Moll and Bläsi, 2002; see Figure 4). Considering the established 70S translation initiation pathway for lmRNAs (Moll et al., 2004; Udagawa et al., 2004), we next tested whether the observed resistance of lmRNA translation to Ksg (Figure 1A) could be attributed to translation initiation of the lmRNA by intact 70S ribosomes. An in vitro translation assay was performed with cross-linked 70S ribosomes, which were previously shown to be competent in translating lmRNA (Moll et al., 2004). In contrast to our expectation, in vitro translation of λ cI mRNA by cross-linked 70S ribosomes was likewise inhibited in the presence of increasing concentrations of the antibiotic (Figure 1C, lanes 2-5), indicating that the observed selective translation of lmRNA in the presence of Ksg in vivo (Figure 1A) can not be attributed to 70S monosomes.
The conflicting in vivo and in vitro results shown in Figures 1A and 1B together with our previous observation that the r-proteins S1 and S2 are dispensable for lmRNA translation (Moll et al., 2002a), prompted us to study whether in vivo translation of cI mRNA is accomplished by a particular population of ribosomes formed in the presence of Ksg. E. coli strain MG1655(pRB381) was grown to an OD600 of 0.3 and then treated with Ksg (750 μg/ml). 60 min upon addition of Ksg the ribosomal profiles were analyzed by sucrose density centrifugation. In contrast to the ribosome profile obtained in the absence of the antibiotic (Figure 2A) no polysomes were detected in the presence of Ksg, i.e. bulk translation was inhibited (Figures 2B and C). Similar to the results by Champney and colleagues obtained with other aminoglycoside antibiotics (Mehta and Champney, 2002; Foster and Champney, 2007), a small 21S peak, corresponding to protein-deficient pre-30S particles accumulated (Figure 2B and C; Figure S2C). During the course of our experiments, we observed a slight shoulder of the 70S peak in ribosome profiles of the control strain MG1655(pRB381) (Figure 2B). However, it was difficult to reproduce the appearance of the shoulder, unless the leaderless cI-lacZ mRNA was simultaneously expressed from plasmid pRB381-1. The presence of the lmRNA resulted in the formation of a distinct 61S peak (Figure 2C). These conditions enabled the purification and biochemical characterization of these 61S particles as specified in Experimental Procedures (Figure S1). The 61S protein composition was determined by Western-blot analysis of the respective particles with antibodies directed against r-proteins (Figure S2B) as well as by mass spectrometry (MS). Both methods gave nearly identical results with the exception of S14 and S20, which were found slightly underrepresented in MS analysis. Furthermore, S15, S17, and S18 were not detectable by MS even in 70S ribosomes. The data are summarized in Figure 2D, showing the protein content of the small subunit of the 61S particles compared to that of the 30S precursor 21S. The 61S particles were deficient in r-proteins S1, S2, S6, S12, S18, and S21, and contained a significantly reduced amount (>50%) of proteins S3, S5, S11, S16 and S17. The group of absent proteins was only partially overlapping with the set of proteins absent in the 21S precursor of the 30S subunit (Figure 2D). In contrast, the protein composition of the 50S subunit was found to be unaltered (data not shown).
Next, we tried to obtain evidence for the functionality of the 61S particles in lmRNA translation in vivo. Protein S12, which was among the proteins absent in the 61S particles, is involved in binding of the antibiotic streptomycin (Ozaki et al., 1969; Carter et al., 2000). We therefore anticipated that 61S particles -if functional- would be resistant to streptomycin (Sharma et al., 2007). As a means to inhibit the translational activity of residual wild-type ribosomes, we added streptomycin to strain MG1655(pRB381-1) after Ksg-induced formation of the 61S particles. In the absence of Ksg and in the presence of streptomycin, a complete shut off of translation was observed (Figure 3A, lanes 2-4). However, when 61S particle formation was induced prior to the addition of streptomycin, the cI-lacZ mRNA continued to be translated in the presence of both drugs (Figure 3A, lanes 5-7) with an efficiency comparable to that seen in the presence of Ksg only (Figure 1A, lanes 5-7). These results indicated that translation of the lmRNA in the presence of both antibiotics, Ksg and streptomycin, is performed by ribosomal particles devoid of S12, and thus most likely by the 61S particles.
In the presence of Ksg no polysomes were detected by densitometry (Figure 2C). However, the 61S particles -if active in lmRNA translation- as well as the lmRNA ought to be present in the polysome fractions. Therefore, we analyzed the corresponding fractions obtained from strain MG1655(pRB381-1) for the presence of the leaderless cI-lacZ and ompA (canonical control) transcripts. After purification of the RNA of the respective fractions of the sucrose gradients (see Figure 2A and C, fractions 11-15) we attempted to detect both mRNAs by primer extension. Both mRNAs were present in the polysome fraction in the absence of Ksg (Figure 3B, lanes 6-10). In contrast, in the presence of Ksg only the cI-lacZ lmRNA (Figure 3B, lanes 1-5) was present in elongating ribosomes. In parallel with the RNA preparation we analyzed the protein composition of the ribosomes present in polysomes of MG1655(pRB381-1) upon Ksg treatment by immunological means. With the exception of proteins S1 and S21, the translationally active ribosomes were devoid of the same set of r-proteins, which were found to be absent in the 61S particles (Figure S2D). For the reasons mentioned in the Discussion, we consider the presence of S1 and S21 in the polysome fraction to be a consequence of direct binding to lmRNA rather than originating from 61S particles.
Using toeprinting, we next tested for the proficiency of the purified 61S particles to form translation initiation complexes at the 5′-terminal AUG of the leaderless λ cI mRNA. As expected from previous results (Moll et al., 2002a; Moll et al., 2004) and the experiments shown in Figure 3B, 70S and 61S particles, respectively, failed to form a translation initiation complex at the canonical ribosome binding site of ompA mRNA (Figure 4B, lanes 2 and 4). In contrast, both the isolated 61S particles and 70S ribosomes formed a translation initiation complex at the 5′-terminal start codon of the λ cI mRNA in the absence (Figure 4A, lanes 2 and 6) and in the presence of Ksg (Figure 4A, lanes 3 and 7).
In addition, in vitro translation assays were performed with an S100 extract containing purified 61S particles, which was programmed with the canonical ompA and the leaderless λ cI mRNA. As expected, the 61S particles were unable to translate the canonical ompA mRNA (not shown). In contrast, the purified 61S particles were functional in translation of the leaderless cI mRNA (Figure 4C, lane 1), and the addition of increasing amounts of Ksg did not affect protein synthesis (Figure 4C, lanes 2 and 3), whereas increasing concentrations of Ksg inhibited in vitro translation in an S100 extract in the presence of added 30S/50S ribosomes (see Figure 1B). Taken the in vivo and in vitro data together, these experiments demonstrated that the 61S particles are functional in translation of lmRNA.
Resistance to Ksg can result from the lack of the di-methylations at two adjacent adenosines at positions 1518 and 1519 in h45 at the 3′-terminus of the 16S rRNA (Figure 5A; Helser et al., 1972; Van Buul et al., 1983). These nucleotides are modified during assembly by the methyltransferase KsgA. We therefore wondered whether the Ksg resistance of the 61S particles is caused by a lack of these methylations. Therefore, primer extension analyses were performed using primer S1525 (Table S1), which binds to the 3′-end of 16S rRNA. Due to the presence of the di-methylations at positions A1518/A1519 in 16S rRNA isolated from MG1655(pRB381-1) 70S ribosomes and 30S subunits, respectively, a stop signal was observed at position 1520 (Figure 5B, lanes 1 and 2). In contrast to the 16S rRNA derived from 21S particles (Figure 5B, lane 4), the 16S rRNA purified from the Ksg-induced 61S particles of strain MG1655(pRB381-1) was likewise modified at the respective positions in h45 (Figure 5B, lane 3). Hence, the resistance of the 61S particle against Ksg could not be attributed to the absence of the methylations at residues A1518/A1519.
To test whether the lack of the r-proteins in the 61S particles can be reconciled with structural changes in the 16S rRNA, we performed in vitro chemical probing experiments using the adenine-specific chemical probe dimethylsulfate (DMS). 70S ribosomes from strain MG1655(pRB381-1) grown in the absence of Ksg (Figure 2A) and 61S particles as well as 70S ribosomes accumulating in the strain treated with Ksg (70SK, Figure 2C) in vivo were purified. The sites of chemical modifications of nucleotides in the 16S rRNA were identified by primer extension analysis using several primers (Table S1). The most striking differences in the modification pattern were observed in the central pseudoknot region, connecting the three major domains of the 16S rRNA (Poot et al., 1996) and h27 (Figure 5A). In contrast to 70S ribosomes, where the nucleotides located in h2 and h27 are protected from DMS modification by base pairing (Figure 5C, lane 10; Figure 5D, black line), we observed an enhanced reactivity of these nucleotides in 70SK ribosomes indicating a disruption of these helices (Figure 5C, lane 2; Figure 5D, red line). The formation of h2 was shown to be essential for binding of proteins S1, S2, S6, S18, and S21 (Poot et al., 1996). It is striking that the same set of proteins is missing in the 61S particles. Nucleotide A908 in 61S particles shows a strong enhancement in DMS reactivity when compared with 70S and 70SK ribosomes (Figure 5C, lane 4; Figure 5D, blue line). This might be attributed to the lack of protein S12, the N-terminal tail of which lies in close proximity to this nucleotide (Lambert et al., 2005).
We observed several stop signals of the reverse transcriptase (RT) in the absence of DMS, which apparently coincide with stable secondary and/or tertiary structures in the 16S rRNA (Figure 5C, lanes 1, 3, and 9). Additional stop signals seen in h26 within 70SK ribosomes as compared with 70S indicate a distortion of this helix (Figure 5C, lane 1 and 9). As shown in Figure 7B, h26 is located on the solvent side of the 30S subunit, where it interacts with the heterodimer S6/S18 (Greuer et al., 1987; Powers and Noller, 1995) and directly contacts protein S1 (Golinska et al., 1981; Sengupta et al., 2001). Again, all three r-proteins are absent in the 61S particles.
The presence of the di-methylations in 16S rRNA of 61S particles indicated that they originated from 70S ribosomes. To test this, we next examined whether the 61S particles can be formed in vitro from 70S ribosomes. The sucrose gradient analysis shown in Figure 6 revealed that incubation of 70S ribosomes with both, Ksg and lmRNA, for 90 min resulted in the formation of 61S particles (red line). In contrast, no 61S particles were formed in the presence of either Ksg (Figure 6, cyan line) or lmRNA (Figure 6, blue line). We also noted that these conditions lead to a partial dissociation of the 70S ribosomes into subunits, which could indicate a destabilization of 70S ribosomes upon addition of either ligand. However, in the absence of Ksg or lmRNA 70S ribosomes remained stable (Figure 6, black line).
Previous in vitro studies have shown that Ksg does not interfere with translation initiation complex formation on lmRNA by 70S ribosomes (Moll and Bläsi, 2002; Schluenzen et al., 2006). As we have shown that Ksg inhibits lmRNA translation by cross-linked 70S ribosomes in vitro (Figure 1C), the initiation inhibitor Ksg seems to affect translation at a post-initiation step, which remains to be elucidated. Surprisingly, translation of lmRNA is performed by 61S particles accumulating in the presence of Ksg in vivo (Figure 2C) and lacking more then six proteins (S1, S2, S6, S12, S18 and S21). Since the modern ribosome can be viewed as a protein-stabilized ribozyme (Steitz, 2008) and most r-proteins are essential (Wilson and Nierhaus, 2005), this observation is unprecedented.
Protein S1 appears to be essential for translation initiation of canonical mRNAs in E. coli, since it facilitates mRNA binding (Boni et al., 1991; Tedin et al., 1997), whereas ribosomes devoid of both S1 and S2 are functional in lmRNA translation (Moll et al., 2002a). Moreover, protein S21, which stimulates the base-pairing potential of the SD-aSD interaction (Backendorf et al., 1981), is essential for translation of MS2 RNA, while the lack of protein S21 did not affect poly(U) translation (van Duin and Wijnands, 1981). Poly(U) resembles lmRNA in that it is translated in vitro under conditions, where 70S monosomes are prevalent (Moll et al., 2004). Collectively, the absence of r-proteins S1, S2 and S21 from 61S particles (Figure 2D) can thus readily explain the strongly diminished translation (initiation) of bulk mRNA (Figure 1A), as well as the deficiency of the 61S particles to form translation initiation complexes on a canonical ribosome binding site (Figure 4B). Likewise, S12 was shown to be dispensable for poly(U) translation (Nomura et al., 1969), and more recently for in vitro translocation (Cukras et al., 2003), again observations which are consistent with the competence of 61S particles to translate lmRNA (Figures (Figures3A3A and and4C4C).
With the exception of S1 and S21, all r-proteins missing in 61S particles were also absent in polysomes translating lmRNA in the presence of Ksg (Figure S2D). Protein S1 has been reported to bind to single-stranded RNA stretches rich in pyrimidines (Draper and von Hippel, 1978), and gel-shift assays showed that free protein S1 binds to the leaderless cI mRNA (I. Moll, unpublished data). In addition, we have recently shown that binding of S1 to the ribosome depends on protein S2 (Moll et al., 2002a), which was absent in the 61S particles (Figure S2D). Likewise, the interaction of protein S21 with mRNA was shown by UV cross-linking (Schouten, 1985). Therefore, we consider the appearance of proteins S1 and S21 in polysomes translating lmRNA in the presence of Ksg to result from direct binding of these proteins to lmRNA.
The nucleotides at positions A1518/A1519 in the 16S rRNA of the 61S particles are di-methylated (Figure 5B). These methylations belong to the small group of universally conserved modifications seen in all kingdoms of life (O'Farell et al., 2004). They are important for a stable folding of the 3′-end of 16S-type rRNA (Micura et al., 2001), suggesting that the conformation of this ultimate stem-loop is crucial for ribosome function (Rife and Moore, 1998; Micura et al., 2001). The pivotal role of the modifications in h45 is supported by the appearance of aberrant ribosomes sedimenting between 70S and 50S as determined by ribosome profile analysis of an E. coli strain deficient for the KsgA methylase, which is responsible for the modifications (data not shown). However, the lack of an active enzyme confers resistance against Ksg (Helser et al., 1972; Van Buul et al., 1983), which might be explained by the direct interaction between h45 and h24, the latter involved in binding of the antibiotic (Schluenzen et al., 2006, Figure 7).
For methylation activity KsgA requires a minimal particle consisting of the 16S rRNA and eight r-proteins (Thammana and Held, 1974; Desai and Rife, 2006). Half of these eight proteins were either absent (S6 and S18) or only present in significantly reduced amounts (S16 and S17; Figure 2D) in the 61S particles. The presence of the methylations in the 61S particles is therefore difficult to reconcile with the assumption that Ksg exerts its effect on an assembly intermediate before binding of the proteins S6, S18, S16, and S17.
Moreover, the structural analysis of the 16S rRNA of 70S ribosomes obtained in the presence of Ksg in vivo (70SK) revealed the disruption of h2 and h27 (Figure 5C, lane 2). These helices together with h24 and h28 form a loop structure (Figure 7A), which entraps the primary binding site of Ksg at the position of the mRNA start codon in the P-site located between G926 of h28 and A792 and A794 of h24 (Woodcock et al., 1991; Schluenzen et al., 2006). Since (i) the presence of a lmRNA is required to stimulate the formation of the 61S particles (Figure 2C and Figure 6), (ii) the formation of a 70S initiation complex on lmRNAs is not inhibited in the presence of Ksg (Figure 4A; Moll and Bläsi, 2002; Schluenzen et al., 2006), and (iii) 61S particles can be formed by incubation of 70S ribosomes with Ksg and lmRNA in vitro (Figure 6) we suggest the following model for in vivo formation of these particles (Figure 7C): (1) 70S ribosomes form an initiation complex exclusively with lmRNA in the presence of Ksg (Figures 4A and B). (2) Ksg changes the 70S conformation, in particular that of the loop-forming helices h2, h27, h24, h28 and h26 (Figures (Figures5C5C and and7A).7A). (3) Disruption of some of these helices (h2, h26, and h27; Figure 5C) triggers the release of ribosomal proteins directly or indirectly attached to this loop.
Since ribosomes of organisms of all three domains can translate lmRNA (Grill et al., 2000; Moll et al., 2002b), they may be viewed as remnants of ancestral mRNAs before domain separation. The 61S particles proficient in lmRNA translation contain a reduced number of 30S proteins. With the exception of S2 and S12, which are conserved in all three evolutionary domains, most of the proteins reduced or absent in 61S particles were most likely added later in evolution, after separation of the kingdoms (Mears et al., 2002). It is therefore tempting to speculate that the 61S particles mirror an ancient form of the small subunit present in proto-ribosomes before domain separation, about three billion years ago.
Cross-linked 70S ribosomes can translate leaderless mRNA (Figure 1C; Moll et al., 2004), and 61S particles are more stable at low Mg2+ concentrations and in the presence of an S100 extract than 70S ribosomes (A. C. Kaberdina and I. Moll, unpublished). It is therefore possible that the 61S particle acts as a stable monosome, and thus resembles the proposed one-subunit ‘proto-ribosome’ that catalyzed peptide bond formation initiating at the 5′-terminus of single-stranded polynucleotides (Moore and Steitz, 2002). Furthermore, the 16S rRNA of 61S particles is methylated at the two residues A1518/1519 (Figure 5A), which is an important feature for stable folding of the 3′-end of 16S-type rRNA (Micura et al., 2001) comprising the decoding center. Since adenine-methylation occurs at this functional hot spot in ribosomes of all three domains, it has probably been acquired at or even before the existence of the bacterial proto-ribosome and thus also before domain separation. We find it a total surprise that under distinct stress conditions ribosome particles are formed in vivo in present bacteria, which might reflect ancient bacterial proto-ribosomes.
E. coli strain MG1655 (Blattner et al., 1997) has been described. Unless otherwise indicated, bacterial cultures were grown in LB broth (Miller, 1972) at 37°C in the presence of 100 μg/ml ampicillin to maintain selection of plasmids pRB381 (Brückner, 1992) and pRB381-1. Growth was monitored by measuring the optical density at 600 nm (OD600). Plasmid pRB381-1 confers ampicillin resistance and harbors a translational fusion in which the first 63 codons of the leaderless λ cI gene are abutted on the eighth codon of the lacZ gene (Moll et al., 2001).
E. coli strain MG1655 (pRB381-1) was grown in M9 minimal medium. At an OD600 of 0.3 either no antibiotic, Ksg (1 mg/ml) or streptomycin (Sm, 100 μg/ml) was added. 30, 60 and 90 minutes after addition of antibiotics 150 μl aliquots were withdrawn from all three cultures. After 60 minutes of incubation with Ksg, Sm was added (100 μg/ml) to half of the culture. 30, 60 and 90 minutes after addition of Sm, aliquots were withdrawn from both cultures (with and without Sm). At each time pulse labeling was carried out by addition of 1.5 μl of L-[U-14C]-aa mix (50 μCi/ml), and by further incubation for 5 min at 37°C. The reactions were stopped by addition of an equal volume of cold 10% TCA followed by incubation on ice for 15 min and subsequent centrifugation for 15 min at 15,000 rpm at 4°C. The cell pellets were washed once with 90% acetone, dried under vacuum for 5 min, resuspended in SDS-protein sample buffer and boiled for 5 min prior to loading onto a 12% SDS polyacrylamide gel. For the different OD600 values, the same amounts of total cellular protein were subjected to electrophoresis. The gels were dried and exposed to a Molecular Dynamics PhosphoImager for visualization.
Full-length λ cI and ompA mRNA (4 pmol) were translated in vitro with E. coli S100 extracts and 30S/50S ribosome, cross-linked 70S ribosomes, or 61S-particles as described before (Moll et al., 2004). Briefly, 15 μl of Mix A (16.6 mM MgOAc, 80 mM NH4Cl, 30 mM Tris-HCl pH 7.7, 3.3 mM DTT, 1.6 mg/ml of E. coli tRNA, 0.2 mM citrovorum, 16.6 mM KCl, 0.33 mM amino acids (-lys), 66.6 μM [14C]-lys, 3.3 mM ATP, 0.66 mM GTP, 16.6 mM phosphoenol-pyruvate and 0.04 mg/ml of pyruvate kinase) were mixed with 5 μl S100 extract and 5 pmol of either 30S/50S ribosomes, cross-linked 70S ribosomes, or 61S particles. The reactions were started by addition of 5 pmol of mRNA. After incubation of the reactions for 30 min at 37°C either in the absence or in the presence of 1.6 μM, 16 μM, 160 μM, or 1.6 mM Ksg, translation was stopped by addition of 4 volumes of 90% acetone. The samples were resuspended in SDS-protein sample buffer and loaded onto a 12% SDS-polyacrylamide gel. Gels were dried and exposed to a Molecular Dynamics PhosphoImager for visualization and quantification.
DMS-modification reactions of the rRNA of purified 70S and 70SK ribosomes and 61S ribosomal particles were performed as described (Stern et al., 1988). Briefly, 2 pmoles of ribosomes or particles were modified in a reaction volume of 45 μl in buffer containing 10 mM Tris-HCl pH7.3, 60 mM NH4Cl, 10 mM MgOAc and 6mM β-mercaptoethanol by addition of DMS (5 μl in a 1/12 dilution in ethanol) followed by incubation on ice for 30 min. Control reactions were performed by adding ethanol without DMS. Reactions were stopped by ethanol precipitation. After phenol/CHCl3 extractions the modified rRNA was resuspended in 24 μl 1× RT-buffer (50 mM Tris-Cl, pH 8.3; 60 mM NaCl; 6 mM MgCl2, 10 mM DTT). For primer extension analysis 2.4 μl (0.2 pmol) of the respective rRNAs were annealed to the 5′-end labeled primers (Table S1; 2.4 μl, 0.6 pmol) in 1× RT-buffer, heated to 80°C for 3 min, snap frozen in liquid nitrogen, and slowly thawed on ice. Primer extension reactions were performed in RT-buffer using the AMV reverse transcriptase (Promega) by incubation at 42°C for 15 min essentially as described before (Moll et al., 2004). The samples were separated on an 8% PAA-8M urea gel and the extension signals were visualized using a Molecular Dynamics PhosphoImager. Quantification was performed using ImageQuant program. To analyze the in vitro DMS modification we used DMS-nonspecific stops as internal standards (G888, U891, G898, C899) to normalize the amounts of the different 16S rRNA.
250 pmol of 70S ribosomes prepared under low salt conditions (60 mM NH4Cl) were incubated in the absence or presence of an equimolar amount of full length cI mRNA in a reaction volume of 100 μl in a buffer containing 10 mM Tris-HCl pH 7.3, 60 mM NH4Cl, 6 mM MgOAc and 6 mM β-mercaptoethanol either in the presence of 25 mM Ksg when indicated. After incubation at 37°C for 90 min the reactions were layered on 7-47% sucrose gradients made up in the reaction buffer and centrifugation was performed as described above.
We are grateful to Dr. U. Bläsi for his constant support, to Drs. B. Redl and J. Rife for antibodies against r-proteins and bacterial strains, and to Konstantin Byrgazov for his help during purification of ribosomal particles. The work was supported by the BMBF-project No. 031 2552 (to K. H. N.) and by an EMBO short term fellowship as well as by grants T259-B11 and P20112-B03 from the Austrian Science Fund (to I. M.).
Experimental Procedures for the preparation of ribosomes, ribosome profile analysis, determination of the protein composition of 61S particles by immunoblotting, detection of mRNAs (cI-lacZ and ompA) in polysomes and toeprinting analysis are described in the Supplementary Material.