The set of oligonucleotides used for primer extension enabled us to scan the complete domain II (positions 644–1457) and the neighborhood of the α-sarcin loop in domain VI (positions 2840–3107) of S.cerevisiae 26S rRNA after chemical modification. Footprinting was performed on ribosomes lacking different stalk ribosomal proteins using intact particles as well as naked 26S rRNA as a control. Specific conformational changes due to the presence of GTPase center proteins were found, but first, some general structural characteristics of the scanned regions will be discussed.
Structural features of the S.cerevisiae 26S rRNA domain II and the α-sarcin region
Primer extensions on naked rRNA usually show a number of reverse transcriptase stops that occurred in all samples. These non-specific stops or ‘control cuts’ correspond to highly stable RNA secondary or tertiary structural features that are maintained at the reverse transcription temperature (26
). Only two strong reverse transcriptase stops, totally blocking the reverse transcriptase activity, have been detected in the tested yeast rRNA regions, corresponding to positions A644 and C2840 (data not shown). Similar stops in eubacterial rRNA have been previously described as being indicative of a base-methylated residue 5′ to the stop. A strong stop at an A644 equivalent position has also been detected in phylogenetically distant organisms such as Drosophila melanogaster
) and the halophilic archaeon Haloferax mediterranei
(C.Briones and R.Amils, unpublished results). These results suggest that methylation at these positions seems to be a conserved feature of the large rRNA.
Comparison of results from chemical modification of naked rRNA and ribosomes showed, as expected, that nucleotides participating in helical regions are protected in both instances. However, stem regions, such as nucleotides 750–781 and 962–966 (Fig. A and B), which are modified in the naked RNA and protected in the ribosome, have also been detected. These double-stranded regions may not be formed in naked RNA, their Watson–Crick interactions being stabilized in the ribosome (see Fig. , for a model of the secondary structure of the studied 26S rRNA regions).
Figure 1 Chemical reactivity of nucleotides around regions 740–790 (A), 950–970 (B), 670–720 (C) and 1330–1370 (D) in S.cerevisiae 26S rRNA domain II from naked RNA (1) and two different ribosome preparations (2,3) tested by primer (more ...)
Figure 2 Secondary structure of the S.cerevisiae 26 rRNA domain II and the α-sarcin region. Summary of nucleotide reactivity changes detected by footprinting is described in the text. Blue circles, higher reactivity in the rRNA than in the ribosome; magenta (more ...)
Almost all of the unpaired nucleotides are reactive in free 26S rRNA but not in the S.cerevisiae
W303 ribosomal particles (Fig. ). Protein–RNA interactions as well as some RNA tertiary structure features protect most of the loop regions from attack by the chemical probes or RNases, as in bacteria (35
) and mammals (36
). In fact, some nucleotides in unpaired regions, like those in the neighborhood of nucleotides 1173–1174 and 1310–1311, correspond to positions predicted to take part in tertiary interactions in eubacteria (37
), suggesting that those long-range interactions also occur in yeast.
A number of nucleotides were found to be more reactive in ribosomes than in naked rRNA. Similar unprotected residues have been also described in E.coli
). They may reflect conformational changes associated with the assembly of the ribosomal particle, or may lie on functional sites of the ribosome, which require unpaired nucleotides to allow the interaction of ligands. The most prominent cases of this type in yeast 26S rRNA domain II are located at A689, A690, A691 (Fig. C), at C716 and A760 in the loop of the variable region V4 (Fig. A and C), at A1224 (Fig. ) and at G1348–A1354 (Fig. D). Moderate stimulations were found in other positions shown in Figure .
Figure 3 Changes in chemical reactivity of nucleotides in the 26S rRNA GTPase domain due to either the absence of acidic ribosomal P1/P2 proteins (A) and protein L12 (B) or the presence of sordarin derivatives (SD) in wild-type ribosomes (C). In (A) and (more ...)
Effect of stalk proteins on GTPase RNA reactivity
The 1218–1224 loop shows highly accessible and neighboring inaccessible nucleotides in S.cerevisiae
ribosomes, following a pattern that was also found in ribosomes from E.coli
). As previously commented, this region corresponds to the binding site of the S.cerevisiae
P0 and associated acidic proteins. The accessibility changes observed in this loop upon protein binding may reflect a way to overexpose some nucleotides involved in functional interactions with translational ligands. The study of the effect caused on the nucleotide reactivity by specific proteins can provide information on their role in this process.
Using gene disruption methods, a set of S.cerevisiae mutants were obtained that lack one or more of the proteins involved in the ribosomal GTPase activity. The results from a footprinting study of the ribosomes from these mutants, summarized in Figure , have yielded information on the RNA regions directly or indirectly affected by the binding of these proteins.
Acidic protein deficient ribosomes
. Saccharomyces cerevisiae
D4567 was deprived of the four stalk acidic proteins, P1α,P1β, P2α and P2β, by gene disruption. The strain is viable in standard conditions but its functional ribosomes, which contain standard amounts of the remaining GTPase proteins, totally lack acidic proteins (16
When the reactivity of the rRNA in ribosomes from wild-type S.cerevisiae W303 and D4567 strains was compared, a number of positions exhibited reproducible accessibility changes. All of them corresponded to a weak reduction of chemical reactivity in the mutant ribosomes. Most of these nucleotides were concentrated in the unpaired regions of the GTPase domain: A1224, G1241, G1242, A1243, A1244, A1269, A1270 and A1272 (Fig. A). Differential signals were also detected in the nucleotides A845, A846, A881 (Fig. A), A1078, A1092, A1101 and A1102 (Fig. B) as well as in the α-sarcin region (G3019, A3024 and G3025) (Fig. C). Most of these nucleotides were unreactive in the naked rRNA and became exposed in the wild-type ribosomes, but they were protected again in ribosomes lacking P1/P2 proteins. However, some of them, such as G1214/2, A1092, A1102 and G3025, which were already exposed in the rRNA, were protected in the defective ribosome as well.
Figure 4 Modification of chemical reactivity in other regions of the 26S rRNA domain II (A and B) and in the α-sarcin domain (C). Effects caused by the presence of acidic P1/P2 proteins, protein L12 and sordarin derivatives (SD) are shown as indicated. (more ...)
These results seem to exclude a direct interaction of the P1/P2 proteins with the rRNA, since in this case the protein removal would be expected to result in an increase of the nucleotide reactivity. They are, however, compatible with an indirect effect on the rRNA conformation induced by the acidic protein binding. Alternatively, since most of the equivalent positions have been shown to be protected by the elongation factors in bacterial ribosomes (39
), and some of them are also protected by EF-2 in mammals (40
), these results might reflect a higher amount of supernatant factors bound to the mutant ribosomes. Supporting this possibility, the differences tend to be reduced by high salt washing, after which the mutant ribosome reactivity is closer to that of the wild-type particles. Since both ribosome preparations are processed and washed in the same way, these results would indicate a higher affinity of the mutant ribosomes for the elongation factors. If so, the presence of the 12 kDa acidic proteins would promote the exchange of the factors in the ribosome rather than facilitate their binding, as has been assumed so far.
A higher affinity of the particles for the supernatant factors due to the absence of the 12 kDa proteins could also provide an explanation for the lower translation efficiency of the mutant particles (16
P0 protein deficient ribosomes
. Protein P0 is essential for ribosome activity and cell viability, but P0-deficient particles can be obtained from a conditional P0 null mutant grown in restrictive conditions for a controlled period of time (30
). These defective particles were used in an attempt to study P0-dependent conformational changes in the GTPase RNA. Unexpectedly, no signal was detected in the footprinting gels when different preparations of these particles were used in primer extension assays (data not shown). Primers designed to anneal into domains I, III, IV and V in the 26S rRNA showed the same negative results. Since the rRNA extracted from the particles prior to treatment did not show substantial differences with the controls these results seem to indicate that RNA degradation must take place during the chemical modification reaction. However, similar negative results were observed when the modification reaction was carried out at either 0°C (26
) or in the presence of diethyl pyrocarbonate to minimize the activity of contaminating RNases.
The apparent high sensitivity of these ribosomes to degradation is exclusively due to the absence of protein P0 since the parental strain, S.cerevisiae W303 and other mutants derived from the same strain, like S.cerevisiae D4567 mentioned formerly, did not present any footprinting problems. It is, therefore, possible that the absence of protein P0 notably increases the susceptibility of rRNA to residual RNase activities in the ribosome preparation. P0 is an essential protein for the GTPase center and its absence could expose a remarkably RNase-sensitive domain in RNA structure.
Protein L12 deficient ribosomes
. Protein L12 has an important role in the structure of the yeast GTPase center, binding to loops 1239–1247 and 1267–1272, which are accessible to chemical probes both in naked rRNA and in ribosomes in agreement with data on E.coli
; C.Briones and R.Amils, unpublished results). These two loops fold over close together in the tertiary structural model of this part of the GTPase domain along with protein L11(L12) (4
) and contribute to the binding site for antibiotics thiostrepton and micrococcin (41
), elongation factors (42
) and rat anti-28S RNA antibody (29
). The bacterial L11 protein is located at the base of the stalk, and in yeast the equivalent protein L12 seems to affect the binding of the P0/P1/P2 complex since some of the stalk components are not found in the ribosome in its absence (22
In order to characterize conformational changes caused by L12 in the RNA, the differential rRNA reactivity patterns of wild-type ribosomes (S.cerevisiae
W303) and particles lacking protein L12 (S.cerevisiae
6EA1) were studied. Two strong protections at G1235 and A1262 and four weaker ones at G1242, A1269, A1270 and A1272 were detected in the presence of protein L12 (Fig. B). G1235 and A1262, corresponding to E.coli
U1061 and A1088, are located in the region protected by the binding of L11 in bacteria (1
). However, while A1088(A1262) forms a conserved long-range base pair with U1060 and interacts directly with protein L11, U1061(G1235) bulges out the helix and seems not to be involved in the protein interaction (Fig. ). Interestingly, nucleotide A1899 in rat 28S rRNA, equivalent to yeast A1262, was also the most protected position upon in vitro
binding of purified rat L12 to naked rRNA. In contrast, the reactivity of the rat equivalent to yeast G1235, nucleotide G1873, was not affected. In its place, A1887 became protected by rat L12 (29
), while the reactivity of the equivalent nucleotide in yeast, A1251, was totally unmodified (Fig. B). These results confirm that the differences between the yeast and rat GTPase center are not limited to the number of different acidic proteins in the stalk, four in the first case and two in the second, but probably extend to structure of the RNA–protein complex. Alternatively, these differences might be due to the fact that in our case native L12-deficient ribosomes were used while an in vitro
reconstituted protein–RNA complex was utilized in the rat system.
Figure 5 Possible secondary structure (A) and two different views of the tertiary structure (B) of the rRNA in the S.cerevisiae GTPase-associated region involved in interaction with protein L12. The structure has been adapted from the model reported for the bacterial (more ...)
The nucleotides showing smaller reactivity changes, G1242, A1269, A1270 and A1272, lie on the terminal loops of the GTPase domain. These two loops come close in the 3-D structure of the L11–RNA complex, but they are not directly involved in the interaction with the protein, although its N-terminal domain is not far from them (5
). In fact, they form together with an L11 proline-rich loop, the binding site of thiostrepton and microccocin, two classic inhibitors of the bacterial GTPase center (43
). Confirming the proximity of L11, protection in both loops by the protein has been reported (1
), and recently they have been targeted by direct hydroxyl probing using Fe(II) tethered to position 19 in L11 (28
Apart from these six nucleotides, the full scanning of the domain II and the α-sarcin region revealed four additional weaker protections in positions A845, A846, A881 and A882 (Fig. A), which coincides with the opposite effect caused by the absence of the acidic P1/P2 proteins as discussed previously. These results suggest that this domain II region lies in the vicinity of the GTPase domain in the 3-D structure of the ribosome and, therefore, collaborates in the GTP-associated reactions involved in the translational process. This implication has not yet been reported in eubacteria, although proximity between both regions of domain II has been proposed based on data from H.mediterranei
ribosome reconstitution experiments (C.Briones and R.Amils, unpublished data). Finally, the region comprising these positions was described as functionally active, being involved in tRNA translocation (44
Taken together, the results confirm the similarity of the L11/L12–RNA complex structure in the different species previously suggested by biochemical data (45
) but at the same time indicate the existence of structural peculiarities in the yeast ribosome that will require the 3-D structure to be established in order to be fully understood.
Effect of GM193663A, a yeast GTPase inhibitor sordarin derivative
The antifungal sordarin derivative GM193663A, has been shown to specifically inhibit yeast protein synthesis by interfering with the interaction of the elongation factor EF-2 with the ribosome (33
). Resistance mutations to sordarin have been found in EF-2 as well as in the stalk protein P0 (10
). A disturbance of the GTPase center by the binding of the inhibitor should, therefore, be expected. To check this possible effect, comparison of rRNA reactivity to chemical probes in S.cerevisiae
W303 ribosomes obtained in the presence and in the absence of the antibiotic was carried out. An increase of the reactivity was detected in nine positions in the GTPase RNA from ribosomes carrying the inhibitor. The strongest stimulation was found in position G1241; a weaker effect was detected in A1224, A1243, A1244, A1269, A1270 and A1272 (Fig. C). Two additional signals were found at the α-sarcin loop in G3019 and G3025 (Fig. C).
As indicated previously, G1241 and A1269 are equivalent to the E.coli
A1067 and A1095. These nucleotides, together with protein L11, are directly involved in the binding of the thiopeptide antibiotics, thiostrepton and microccocin (43
). Like sordarin derivatives in yeast, these antibiotics block the function of the bacterial elongation factor EF-G. It may be tempting to conclude from our footprinting results that GM193663A acts in the yeast ribosome in a similar way to thiopeptide antibiotics in eubacteria, especially considering that microccocin, like the sordarin derivatives, stimulates the reactivity of A1067 (27
). In fact, although the available information indicates that sordarin derivatives interact with the elongation factor (48
), the resistance mutations found in protein P0 (10
) indicate that the stalk structure must also be affected exposing some nucleotides, as shown in this report. Moreover, as previously commented on, these nucleotides are among those involved in elongation factor interaction, in agreement with the inhibition of EF-2 function by the sordarin derivatives. Interestingly, the EF-G inhibitor fusidic acid has an opposite effect to sordarins, protecting rather than exposing equivalent nucleotides in eubacterial ribosomes. It seems, therefore, that both types of compound act in a different way in agreement with the biochemical data (49