Tet(○) is an elongation factor-like protein found in clinical isolates of Campylobacter jejuni that confers resistance to the protein-synthesis inhibitor tetracycline. Tet(○) interacts with the 70S ribosome and promotes the release of bound tetracycline, however, as shown here, it does not form the same functional interaction with the 30S subunit. Chemical probing demonstrates that Tet(○) changes the reactivity of the 16S rRNA to dimethyl sulphate (DMS). These changes cluster within the decoding site, where C1214 is protected and A1408 is enhanced to DMS reactivity. C1214 is close to, but does not overlap, the primary tetracycline-binding site, whereas A1408 is in a region distinct from the Tet(○) binding site visualized by cryo-EM, indicating that Tet(○) induces long-range rearrangements that may mediate tetracycline resistance. Tetracycline enhances C1054 to DMS modification but this enhancement is inhibited in the presence of Tet(○) unlike the tetracycline-dependent protection of A892 which is unaffected by Tet(○). C1054 is part of the primary binding site of tetracycline and A892 is part of the secondary binding site. Therefore, the results for the first time demonstrate that the primary tetracycline binding site is correlated with tetracycline's inhibitory effect on protein synthesis.
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The widespread use of tetracycline for over 50 years in both medicine and as an animal growth promoter has increased the occurrence of tetracycline resistance in microbial organisms (reviewed in Chopra and Roberts, 2001). Efflux and ribosomal protection are by far the most common resistance determinants and are found widely in both Gram-positive and -negative species (Chopra and Roberts, 2001). The dissemination of these resistance determinants is largely dependent on horizontal gene transfer events (Chopra and Roberts, 2001). Efflux mechanisms limit the accumulation of toxic tetracycline levels in the cytoplasm. In contrast, the ribosomal protection proteins (RPPs) interact directly with the target of tetracycline, the ribosome, to promote release of the bound drug. Two less common resistance mechanisms are chemical modification of tetracycline (Speer and Salyers, 1989) and mutations in the 16S rRNA genes [seen in Helicobacter pylori and Propionibacterium acnes (Ross et al., 1998; Trieber and Taylor, 2002)].
The role and interaction of the RPPs with the 70S ribosome during tetracycline release has been studied primarily using the RPP determinants Tet(○) and Tet(M). These specific determinants exemplify the RPPs but this class of proteins also includes Tet(Q), Tet(S), Tet(W) and OtrA. Tet(○) and Tet(M) are generally found on mobile genetic elements, which explains their wide dissemination throughout the eubacteria (Chopra and Roberts, 2001). The RPPs appear to be derived from Gram-positive species possibly evolving from OtrA (Sougakoff et al., 1987; Taylor, 1992), which is found in the natural producer of oxytetracycline, Streptomyces rimosus (Doyle et al., 1991). Sequence comparisons show that the amino-terminal GTP binding domains (G-domain) of the RPPs are very similar to the G-domains of other ribosome binding proteins such as the elongation factors and, in the case of EF-G, this sequence similarity exists throughout the length of the protein, albeit to a lesser degree (Sanchez-Pescador et al., 1988; Manavathu et al., 1990; Burdett, 1991). This similarity extends to the three-dimensional structure as a 16 Å resolution cryo-EM reconstruction demonstrated that Tet(○) and EF-G have an overall similar shape (Spahn et al., 2001). The similarity of the RPPs’ G-domain to that of the elongation factors is exemplified by the fact that a mutation which changes EF-Tu from a GTPase to a XTPase (Hwang and Miller, 1987; Weijland et al., 1994) can also be made in Tet(○) on the basis of sequence alignments alone (C.A. Trieber, unpublished data). Furthermore, like the elongation factors, the RPPs require GTP to function (Grewal et al., 1993; Burdett, 1996) and Tet(M) has been shown to compete with EF-G for binding to the ribosome (Dantley et al., 1998). However, the RPP is not functionally equivalent to the elongation factors in vivo or in in vitro assays (Burdett, 1991; 1996).
Crystallographic studies have revealed several tetracycline binding sites on the 30S ribosomal subunit (Brodersen et al., 2000; Pioletti et al., 2001). The most highly occupied (primary) site is located such that it sterically interferes with accommodation of the tRNA in the A-site (Brodersen et al., 2000; Pioletti et al., 2001). This agrees well with early studies showing that tetracycline functions as an inhibitor of aminoacyl-tRNA binding to the A-site (Hierowski, 1965; Suarez and Nathans, 1965; Lucas-Lenard and Haenni, 1967). In the primary binding site, tetracycline interacts with the irregular minor grove of helix 34 (h34; rRNA residues 1196–1200, 1053–1056) and the loop of helix 31 (h31; rRNA residues 964–967) (Brodersen et al., 2000; Pioletti et al., 2001). Upon tetracycline binding, Brodersen and colleagues state that the ribosome appears to be competent for the initial stages of ternary complex (EF-Tu•GTPaa•tRNA) binding, namely decoding and GTP hydrolysis (Gordon, 1969; Brodersen et al., 2000). The subsequent accommodation step of the tRNA into the 50S A-site, however, is effectively blocked preventing the extension of the nascent chain. The ribosome may then be locked in a non-productive and energetically expensive cycle of ternary complex binding and release (Brodersen et al., 2000). The RPPs would be expected to interfere with this unproductive cycle by binding to the tetracycline-blocked ribosome, triggering the release of tetracycline and returning it to the elongation cycle (Spahn et al., 2001).
Visualization of Tet(○) on the Escherichia coli 70S ribosome (16 Å resolution) showed that Tet(○) binds to the ribosome in a similar fashion to the elongation factors (Spahn et al., 2001). In this position, Tet(○) approaches the 70S ribosome from the A-site side and binds in the intersubunit space contacting the 50S subunit near the base of L7/L12 stalk in the vicinity of both the α-sarcin/ricin loop (H95) and thiostrepton/L11 binding site (H43/44) (Spahn et al., 2001) (throughout this manuscript, helix abbreviated with a lower case ‘h’ refers to a helix within the 16S rRNA, whereas an upper case ‘H’ refers to one within the 23S rRNA). On the 30S subunit, Tet(○) contacts the ribosomal protein S12, and the 16S rRNA at h5 and h34 of the decoding site (Spahn et al., 2001). Despite the general similarity between the interaction of Tet(○) and EF-G with the ribosome, Tet(○) is unable to invoke any of the gross conformational changes seen with EF-G (Frank and Agrawal, 2000) with the exception of the extension of the L7/L12 stalk (Spahn et al., 2001). When the high-resolution X-ray structures depicting a tetracycline–ribosome complex (Brodersen et al., 2000) are combined with the cryo-EM maps of the 70S•Tet(○) complex, Tet(○) appears to approach, but does not overlap the primary tetracycline binding site (Spahn et al., 2001). Domain IV of Tet(○) instead contacts the base of h34 (Spahn et al., 2001).
In this study, we investigated the interaction of Tet(○) with the 16S rRNA component of the 70S ribosome by chemical probing. This method monitors the chemical accessibility of the rRNA bases and is therefore sensitive to subtle architectural changes, which might go unnoticed by cryo-EM or be constrained by crystal packing forces in the high-resolution X-ray crystallography structures. We identified specific sites of Tet(○) interaction with the 16S rRNA that are suggestive of both a close contact and of long-range conformational rearrangements. These interactions may form the basis of Tet(○)-mediated tetracycline resistance.
Tet(○)GMPPNP binding to the 70S ribosome
To establish the conditions needed to bind Tet(○) to the ribosome or ribosomal subunits, we measured the binding of tetracycline when Tet(○) was added in the presence of GMPPNP. It was shown previously that Tet(○) in the presence of GTP or a non-hydrolysable GTP analogue (GMPPNP or GTPγS) was able to remove tetracycline from the 70S ribosome but, only in the presence of GTP, was Tet(○) able to act in multiple turnover experiments (Trieber et al., 1998). When the non-hydrolysable analogues were used, an excess of Tet(○) over ribosomes was required to remove tetracycline, suggesting that without GTP hydrolysis Tet(○) lost its ability to recycle and was locked in a stable complex with the ribosome (Trieber et al., 1998). In this case the binding of Tet(○) is irreversible, tetracycline is prevented from rebinding and therefore Tet(○) binding can be measured indirectly by following tetracycline release. In addition, as tetracycline binds similarly to both 30S subunits and 70S ribosomes, we also tested the ability of Tet(○) to remove tetracycline from the 30S subunit.
The 70S ribosomes or 30S subunits were incubated with tetracycline to bind the drug and subsequently, Tet(○) and GMPPNP were added to the mixture and the incubation continued. As the concentration of Tet(○) increases with respect to the ribosome concentration, the relative tetracycline binding to the 70S ribosomes decreases until reaching a minimum when Tet(○) was present in a three molar excess over ribosomes (Fig. 1). Increasing the Tet(○) concentration above this point did not further the removal of tetracycline, suggesting that tetracycline was occupying sites that were not accessible to be released by Tet(○).
Because it has been suggested that the inhibitory tetracycline binding site is located on the 30S subunit (Goldman et al., 1983; Brodersen et al., 2000; Pioletti et al., 2001), we investigated the ability of Tet(○) to act on the 30S subunit alone. In the concentration range where Tet(○) was able to fully remove tetracycline from 70S ribosomes, it had no effect on tetracycline binding to 30S subunits (Fig. 1), suggesting that Tet(○) is unable to form a functional and stable interaction with the 30S subunit alone.
Interaction of Tet(○) with the 16S rRNA
Contact between Tet(○) and the ribosome is generally mediated by the rRNA, with the exception of a single contact between domain III of Tet(○) and the ribosomal protein S12 (Spahn et al., 2001). To study the interaction of Tet(○) with the 30S ribosomal subunit, the target of tetracycline, we monitored the change in dimethyl sulphate (DMS) reactivity of the 16S rRNA in response to Tet(○) binding. For these experiments Tet(○), EF-G and tetracycline were bound to 70S·poly(U)·AcPhe-tRNAphe complexes in the presence of GMPPNP. In this complex, AcPhe-tRNAphe is bound in an mRNA-dependent manner to the P-site. After preparation, the complexes were treated with DMS, which modifies adenosine at its N1 position and cytosine at its N3 position, depending on their accessibility and pKa values. Using primer extension analysis, we identified two Tet(○)-dependent changes in DMS reactivity, one at the base of h34 and one within h44 which, surprisingly, is not structurally associated with tetracycline binding (Brodersen et al., 2000; Pioletti et al., 2001). No other bases in the 16S rRNA experienced changes in chemical reactivity upon Tet(○) binding. More then 90% of the 16S rRNA was scanned, with the exception of the initial 20 bases at the 5′-end, and 100 bases at the 3′-end.
Typical gels illustrating the pattern of reverse transcriptions stops in h34 and h44 are shown in Fig. 2. In h44, Tet(○) enhances DMS modification of A1408, which is reflected in Fig. 2A, lanes 2 and 4, as a marked increase in the amount of product formed by blockage of the reverse transcriptase at the 3′-base, C1409, when compared with lane 1. Figure 2B (lanes 2 and 4) shows a clear difference in the pattern of reverse transcription stops, where at the base of h34, at the junction of h32, 33 and 34, Tet(○) protects C1214 from DMS modification. In Table 1, the intensity of the bands that undergo changes in accessibility upon Tet(○) binding have been quantitated (see Experimental procedures) and expressed as a ratio compared with the corresponding band in the unbound 70S·poly(U)·AcPhe-tRNAphe complex.
Table 1. . Tet(○)- and EF-G-dependent alterations in 16S rRNA DMS reactivity.
Tet(○) was bound to the 70S·poly(U)·AcPhe-tRNAphe complex both in the presence and absence of 250 µM tetracycline. Comparison of lanes 2 and 4 in Fig. 2 clearly shows that the presence of tetracycline does not have a gross affect on the Tet(○)-dependent protection of C1214 and enhancement of A1408 toward DMS modification. Quantification of C1214 (Table 1) indicates that its relative protection is decreased in the presence of 250 µM tetracycline possibly as a consequence of a competition between Tet(○) and tetracycline. This trend though is not observed in the case of the enhancement of A1408 to DMS modification (Table 1). It could, however, be obscured by the high variability in the relative enhancement of A1408 in the presence of Tet(○) and tetracycline. Interestingly, EF-G also appears to enhance the reactivity of C1400 and A1408 to DMS (Fig. 2A, lane 5; Table 1). The increase in DMS reactivity of A1408 and C1400 in the presence of EF-G has not been previously reported in the literature to our knowledge but has been observed in a 70S·EF-G·fusidic acid complex (K. Wilson, personal communication).
The bases that undergo changes in DMS reactivity in a Tet(○)-dependent manner are located in the decoding site, a location where possibly mRNA or tRNA could influence the accessibility of DMS. Tet(○), tetracycline, and EF-G had no affect on the level of [14C]-AcPhe-tRNAphe binding to the ribosome compared with the 70S·poly(U) ·AcPhe-tRNAphe complex alone (data not shown), thus eliminating the possibility that differences in the tRNA occupation contributed to alterations in the DMS modification described below. To determine if the reported changes in DMS reactivity were only dependent on the protein, we bound Tet(○) to empty ribosomes and ribosomes complexed with tRNA and/or poly(U) mRNA. In all cases, the characteristic C1214 protection and A1408 enhancement were clearly visible (Fig. 3A and B), indicating that they are a consequence of the interaction between Tet(○) and the ribosome. In addition, heat inactivation (95°C, 10 min) of Tet(○), before addition to the 70S ribosome eliminated both the protection and the enhancement, demonstrating that they are due to the protein and not an unknown component of the buffer system (data not shown).
Influence of Tet(○) on tetracycline-dependent alterations in DMS modification
The interaction of tetracycline with 16S rRNA has been studied previously by chemical probing where it was shown to protect A892 and enhance C1054 and U1052 towards DMS modification (Moazed and Noller, 1987). In this study, these three sites of interaction were confirmed (Fig. 4A; lane 3) such that 250 µM tetracycline was able to decrease the modification of A892 by a factor of two, and increase the modification of C1054 and U1052 by roughly the same (Table 2). As stated above, DMS usually modifies only adenosine and cytidine at neutral pH in a manner that can be detected by primer extension analysis, thus the detection of DMS-modified uridine is uncommon but observed (Moazed and Noller, 1987; Bayfield et al., 2001) and may reflect that U1052 is in an unusual chemical environment. When bound to the 70S·poly(U)·AcPhe-tRNAphe complex in the absence of tetracycline (Fig. 4A; lane 2), Tet(○) had no significant effect on the DMS modification of the bases implicated in drug binding. This suggests that Tet(○) does not interact with the sites of tetracycline binding directly in a manner that can be detected by DMS probing or that it does not interact with these sites in the absence of tetracycline. When Tet(○) is bound in the presence of 250 µM tetracycline and compared with a control complex with only tetracycline bound (Fig. 4A; lanes 3 and 4) a weak but reproducible protection of C1054 by Tet(○) was observed. This decrease in DMS reactivity was observed in all cases but quantification of the data was not conclusive when the relative modification of C1054 was averaged and the error considered (Table 2).
Table 2. . Effect of Tet(○) on tetracycline-dependent alterations in DMS modification.
In Figure 4B and C, the effect of Tet(○) on tetracycline-dependent alterations in DMS reactivity over a range of drug concentrations (2.5–250 µM) was investigated and the results are plotted (Fig. 4D and E). At lower concentrations of tetracycline, the effect of Tet(○) on the DMS modification of C1054 is more apparent. In Fig. 4C, C1054 and U1052 show visibly enhanced modification when tetracycline is present in concentrations greater then 25 µM, whereas at the same concentrations, but in the presence of Tet(○), the enhancement is clearly reduced. This indicates that Tet(○) is responsible for the decrease in DMS reactivity of C1054 and U1052 in the presence of tetracycline. Unlike C1054, the protection of A892 from DMS modification is largely unaffected by Tet(○) (Fig. 4B). At concentrations above 100 µM, the protection of A892 is observed both in the presence and absence of Tet(○). In Fig 4D and E, where the relative modification of A892 and C1054 has been plotted as a function of the tetracycline concentration, it can be seen that over the range of tetracycline concentrations Tet(○) inhibits the tetracycline induced increase of DMS modification at C1054, although having no effect on A892. The low level modification of C1054 in the presence of Tet(○) could be a consequence of incomplete occupation of the ribosome with Tet(○). This effect is shown in Fig. 1, where in the presence of excess Tet(○), some ribosomes are still bound by tetracycline. In this case, modification of C1054, to a small degree, would occur regardless of the presence of Tet(○) as seen in Fig. 4D. These results suggest that Tet(○) prevents the interaction of tetracycline with C1054 while ignoring tetracycline bound to A892.
In many pathogenic bacteria, tetracycline resistance is becoming increasingly common and in the case of the intestinal pathogen, Campylobacter jejuni, tetracycline resistance is conferred by RPP called Tet(○). Recent cryo-EM analysis of a 70S·Tet(○)·GTPγS complex indicates that Tet(○) binds to the elongation factor binding site on the 70S ribosome but does not overlap the primary tetracycline binding site (Spahn et al., 2001). Using DMS probing, we have identified two sites of interaction between Tet(○) and the 16S rRNA; nucleotides C1214 and A1408 (Fig. 5A). The contacts with the 30S subunit alone are not enough to promote binding and/or a functional interaction with the 30S subunit, as we have shown that Tet(○) cannot release tetracycline from the 30S subunit but only from the intact 70S ribosome (Fig. 1). Additionally, by following the tetracycline-dependent changes in DMS reactivity, we have shown that Tet(○) prevents the interaction of the drug with C1054 and U1052 but not A892 (Fig. 4).
Recent structural studies suggest that these changes in DMS accessibility of C1054 and A892 correspond to tetracycline binding to two discrete sites on the 30S subunit; the primary (h34 and h31) and secondary (h27 and h11) sites (Brodersen et al., 2000; Pioletti et al., 2001). In the secondary binding site, tetracycline interacts directly with A892:N1, which explains the protection of this position, whereas in the primary binding site a slight shift of C1054 can explain the increase in DMS modification of this base (Brodersen et al., 2000; Pioletti et al., 2001). The enhanced modification of C1054 and U1052 at low concentrations of tetracycline (Fig. 4D) is in agreement with the dissociation constant (Kd = 2–20 µM) for the single, high affinity, inhibitory tetracycline binding site (Goldman et al., 1983 and references within). The protection of A892 both in the presence and absence of Tet(○) suggests that Tet(○)′s function is not the removal of tetracycline from the secondary binding site but rather to release tetracycline specifically from the primary binding site. This and the clustering of the Tet(○)-dependent alterations in DMS reactivity near the A-site suggest that the primary tetracycline binding site is the inhibitory site and the site that Tet(○) serves to clear (Fig. 5C). It should be noted, however, that the functional significance of the other four tetracycline binding sites observed by Pioletti and colleagues (Pioletti et al., 2001) cannot be assessed as we were not able to follow their release by DMS probing although they are not correlated with the sites that experience changes in DMS reactivity upon Tet(○) binding.
The localization of a Tet(○)-dependent protection in the base of h34 (Fig. 5A) is in strong agreement with the model of Tet(○) binding demonstrated by cryo-EM (Spahn et al., 2001), and is indicative of a close association of Tet(○) with C1214. The protection of a base from chemical modification generally indicates that the base is being shielded directly or that it undergoes a conformational change resulting in decreased accessibility. Figure 5C illustrates that the cryo-EM derived density of Tet(○) (red) (Spahn et al., 2001) is in close proximity to C1214 (cyan wire frame), and could be interacting directly via unresolved elements or alternatively indirectly though other contacts in h34 (blue ribbon). This is in contrast to the A1408 (orange wire frame) enhancement in h44 (red ribbon) because an enhancement results entirely from a conformational change that increases the chemical accessibility of a base. In this case, Tet(○) could generate a rearrangement in h44 without close contact, but rather via indirect interactions possibly using ribosomal protein S12 as a intermediary. In accordance with this notion, the core of S12 makes contact with the backbone of h44 around residues 1491 and 1492 (Brodersen et al., 2002) and cryo-EM reconstructions show that Tet(○) does not approach h44 (Fig. 5C; red ribbon) but does contact S12 (Spahn et al., 2001).
Out of the three sites shown on the 16S rRNA secondary structure in Fig. 5(A), that undergo alterations in DMS reactivity upon EF-G or Tet(○) binding, the C1400 enhancement (violet circle) is characteristic of EF-G, the A1408 enhancement (orange circle) is common to both whereas only the C1214 protection (blue circle) is unique to Tet(○), suggesting the importance of the h34 interaction in Tet(○) activity. Although a C1214 interaction seems to be unique in the literature to Tet(○), the role of helix 34 in ribosomal activity is quite well documented. Mutational analysis of h34 has shown that it is involved in maintenance of translational fidelity particularly in stop codon decoding and frameshifting (Moine and Dahlberg, 1994). The importance of this helix is confirmed by the fact that it is targeted by two antibiotics, tetracycline (Brodersen et al., 2000; Pioletti et al., 2001) and spectinomycin (Carter et al., 2000), which block A-site occupation and inhibit translocation respectively. A role for h34 in translocation has also been suggested recently by chemical probing experiments showing C1054, A1201 and C1203 are protected when EF-G is bound to a pretranslocational complex stalled with thiostrepton (Matassova et al., 2001). The EF-G-dependent DMS protections reported here and by Matassova and colleagues probably differ because of the nature of the ribosomal complex used. Matassova and colleagues propose that the protections induced by EF-G are indirect and are a consequence of a conformational change in h34 that destabilizes the interaction between h34 and the A-site bound tRNA thus facilitating translocation (Matassova et al., 2001). This dynamic nature of h34 is in agreement with that envisioned for the role of h34 in tetracycline resistance, where the direct interaction of Tet(○) with the base of h34 would induce a local disturbance in h34 disrupting the binding pocket of tetracycline.
Unlike C1214, which seems to have a unique interaction with Tet(○), A1408 has been shown to undergo changes in DMS accessibility when the ribosome is bound to various factors. The binding of the IF1 (Dahlquist and Puglisi, 2000), EF-G (this study), Tet(○) (this study) and the translocation inhibitor hygromycin (Moazed and Noller, 1987) all enhance the reactivity of A1408 whereas A-site bound tRNA (Moazed and Noller, 1990) and aminoglycoside antibiotics like paromomycin (Moazed and Noller, 1987) protect it. In some part, these changes can be explained by the dynamic nature of the internal loop of h44 containing A1408. In this loop, A1492 and A1493, the latter of which is basepaired with A1408 (Fourmy et al., 1996), flip out of h44 and insert into the minor groove of the helix formed by the interaction of the codon (on the mRNA) and anticodon (on the tRNA) during decoding (Ogle et al., 2001). Analysis of cryo-EM maps of EF-G bound to the ribosome suggests that domain IV of EF-G is contacting h44 and that it distorts the upper half of this helix during the course of translocation (VanLoock et al., 2000), possibly leading to the enhancement of DMS modification of C1400 and A1408.
The common effect of Tet(○) and EF-G on A1408 of h44 could result from the fact that Tet(○) is evolutionarily derived from EF–G and the interaction of Tet(○) with h44 may simply be a consequence of their common ancestry. On the other hand, tetracycline seems to influence h44 such that its binding disrupts a C967-C1400 UV-induced crosslink and enhances a C1402-C1501 UV-induced crosslink (Noah et al., 1999). Additional evidence that tetracycline may alter h44 derives from the fact that both streptomycin and tetracycline are able to block the cleavage between nucleotide 1493 and 1494 by colicin E3 (Dahlberg et al., 1973). In this sense, the interaction of Tet(○) with h44 might not be an evolutionary relic but rather could be important in either the release of tetracycline directly or by counteracting effects induced by tetracycline in h44.
Our results show that Tet(○) interacts with the base of h34, releases tetracycline from the primary binding pocket and makes further long-range conformational changes in h44, altering the decoding site. The localization of these contacts and/or conformational changes to regions implicated in tetracycline binding and activity suggest they contribute to Tet(○)-mediated resistance to tetracycline.
Determination of Tet(○)-dependent tetracycline release
The ability of Tet(○) to chase tetracycline from the ribosome was determined using a modification of the tetracycline binding assay described earlier (Trieber et al., 1998). Tetracycline was bound to 70S ribosomes or 30S subunits in a mixture containing 2 µM 70S ribosomes (or 30S subunits), 10 µM [3H]-tetracycline (NEN; 100 dpm/pmol), 0–10 µM purified Tet(○), and 50 µM GMPPNP (Roche) in 12.5 µl binding buffer (20 mM Hepes-KOH [pH 7.5], 6 mM Mg acetate, 150 mM NH4 acetate, 4 mM β-mercaptoethanol, 2 mM spermidine and 0.05 mM spermine) and incubated for 10 min at 37°C. The binding reaction was then vacuum-filtered through a 0.45 µm nitrocellulose filter and washed twice with 2 ml of binding buffer to remove the free [3H]-tetracycline. The ribosome-bound [3H]-tetracycline was then quantitated by scintillation counting.
Preparation and modification of complexes
Reassociated E. coli 70S ribosomes from E. coli strain Can 20 were prepared as described earlier (Blaha et al., 2000). Preparation of AcPhe-tRNAPhe followed previously established methods (Rheinberger et al., 1988). The protein factors were complexed with reassociated 70S ribosomes programmed with poly(U) mRNA (Amersham Biosciences) and P-site-bound AcPhe-tRNAPhe. Initially 50 pmols of reassociated 70S was incubated for 15 min at 37°C with 40 pmols of AcPhe-tRNAPhe, and 50 µg of poly(U) in 50 µl of N10 buffer (20 mM Hepes-KOH, pH 7.5; 10 mM Mg acetate, 100 mM NH4 acetate, and 4 mM β-mercaptoethanol) to fill the P-site with tRNA. Subsequently 200 pmols of purified protein factor [EF-G or Tet(○)] and 5 nmols of GMPPNP in 50 µl of N10 buffer were added to the programmed 70S ribosomes and incubated for a further 30 min at 37°C. Tet(○) was added at a 4:1 ratio (protein to ribosome), because previous experiments showed that this was sufficient to achieve a high occupancy of Tet(○) on the ribosome (Fig. 1). After the 37°C incubation, 5 µl of the complexes was taken for nitrocellulose filter binding tests to determine the level of tRNA binding in the complex, which was unaffected by the presence of EF-G or Tet(○) (0.7–0.8 [14C]-AcPhe-tRNAPhe per 70S ribosome). The remaining 95 µl of the 70S ribosome complexes was chemically modified by adding 1.9 µl of dimethyl sulphate (DMS) diluted (1:5) in ethanol (or only ethanol to the unmodified control) and incubated for a further 10 min. The modification reaction was stopped by the addition of 25 µl of DMS stop buffer (1 M Tris-HCl, pH 7.5; 1 M β-mercaptoethanol, 0.1 M EDTA) and 300 µl of 95% ethanol. The rRNA was precipitated and resuspended in 200 µl of TE/SDS (10 mM Tris-HCl, pH 7.5; 100 mM NaCl, 0.5% SDS, 5 mM EDTA) and phenol extracted three times (one volume), followed by three chloroform extractions (one volume). Finally, the rRNA was ethanol precipitated and resuspended in water to a final concentration of 0.1 µg µl−1.
Primer extension analysis
Dimethyl sulphate modification of adenosine and cytosine stops cDNA synthesis by the reverse transcriptase one base before the site of modification allowing for the localization of modification by primer extension experiments. Primer extension analysis of the modified isolated rRNA was performed as described (Polacek and Barta, 1998) using the primers described previously (Moazed et al., 1986). In total, 0.6 pmol of the appropriate primer, 32P-labelled at the 5′-end, was hybridized to approximately 1 pmol of rRNA, which served as the template for primer extension. The extension reaction was carried out using 0.4 units of AMV reverse transcriptase (Roche) for 45 min at 42°C in a buffer containing 122.5 mM Tris-HCl [pH 8.4], 11 mM MgCl2, 15 mM KCl, 11 mM DTT, 250 µM dNTPs. For sequencing reactions, dideoxynucleotides were added to a concentration of 5 µM. The cDNA products of the primer extension reaction were ethanol-precipitated and loaded on a 6% polyacrylamide gel. Gels were scanned using a Molecular Dynamics Phosphorimager and quantitated using the imagequant software package (Molecular Dynamics). Changes in the DMS modification profile were identified by visual inspection and, using an area profile of each lane, normalized according to bands corresponding to DMS independent stops. Bands that displayed visible changes in the phosphorimager scans were quantitated by integrating the area under the peak in the normalized area profile. Reported alterations were visible in multiple primer extension experiments performed on independent complexes, where the values reported correspond to the average and standard deviation of all experiments.
We would like to extend our thanks to P. M. Fucini, and D. Wilson for support and helpful discussions, and to C. M. T. Spahn for providing the cryo-EM map of Tet(○) bound to the 70S ribosome. In addition, we are grateful to K. Wilson, D. Tracz, T. Lawley, M. Gilmour, J. Gunton and N. Polacek for critical review of this manuscript. This work was funded by the Alberta Heritage Foundation through an AHFMR Studentship to S.R.C. and an AHFMR Scientist Award to D.E.T., a grant from the National Science and Engineering Research Council of Canada (NSERC) to D.E.T. and a grant from the Deutsche Forschungsgemeinschaft to K.H.N. (Ni174/8–3).