The A2453-C2499 wobble base pair in Escherichia coli 23S ribosomal RNA
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《核酸研究医学期刊》
Department of Molecular Biology, Cellular Biology and Biochemistry, Brown University, Providence, RI 02912, USA
* To whom correspondence should be addressed. Tel: +1 401 863 2223; Fax: +1 401 863 1182; Email: Albert_Dahlberg@Brown.edu
Present address: Mark A. Bayfield, NICHD, NIH, Bethesda, MD 02892, USA
ABSTRACT
Peptide bond formation, catalyzed by the ribosomal peptidyltransferase, has long been known to be sensitive to monovalent cation concentrations and pH. More recently, we and others have shown that residue A2451 in the peptidyltransferase center of the Escherichia coli 50S ribosomal subunit changes conformation in response to alterations in pH, depending on ionic conditions and temperature. Two wobble pairs, A2453-C2499 and A2450-C2063, have been proposed as potential candidates to convey pH-dependent flexibility to the peptidyltransferase center. Each is presumed to possess a near-neutral pKa, and both lie in proximity to A2451. We show through mutagenesis and chemical probing that the identity of the A2453-C2499 base pair, but not the A2450-C2063 base pair, is critical for the pH-dependent structural rearrangement of A2451. We conclude that, while the A2453-C2499 base pair may be important for maintaining the structure of the active site in the E.coli peptidyltransferase center, its lack of conservation makes it, and consequently its near-neutral pKa, unlikely to contribute to function during peptide bond formation.
INTRODUCTION
For four decades, the mechanism by which the ribosome catalyzes peptide bond formation has been sought. Early works focused on attempts to determine which ribosomal protein (or set of proteins) constituted the peptidyltransferase enzyme, in concert with the then understanding that all enzymes were proteins. Accordingly, typical properties of the enzyme were assessed, e.g. its dependence upon magnesium ions and its pH profile (1). Frustratingly, although a number of proteins clearly enhanced peptide bond formation (2), none in isolation was shown capable of catalysis (3). The discovery of catalytic RNA in the 1980s (4) altered the direction of research. Early insights (5,6) into the evolution of the translation machinery were re-evaluated and remarkable attempts were made to establish that RNA alone, specifically 23S RNA, was capable of catalytic function (7–9). Not until the solution of the crystal structure of the Haloarcula marismortui large ribosomal subunit was achieved, however, did it become absolutely clear that the peptidyltransferase center was completely devoid of protein (10). Since then, the attention has focused almost exclusively on attempts to determine which RNA residue(s) constitute the active site.
The investigation of the ionic requirements of the peptidyltransferase reaction revealed that the active site was conformationally labile. The removal of monovalent cations, in particular, resulted in ribosomes that were incapable of forming peptide bonds (as measured by the puromycin reaction). Inactivation was not irreversible, however; activity could be restored by adding back monovalent cations but a heating step was also required (11), presumably to help re-establish the native, active conformation. We (12) recapitulated these earlier findings and showed that the accessibility of residues in the active site and elsewhere in the ribosome to modification by dimethyl sulfate (DMS) was correlated with the active/inactive transition. At the same time, we demonstrated that there was a change in reactivity of certain residues to DMS that was dependent upon pH, and which also correlated with the active/inactive transition.
The sharp pH dependence of the peptidyltransferase reaction, regardless of whether the substrate for the puromycin reaction was full-length tRNA or the terminal CCA T1 ribonuclease fragment, suggested participation of a functional group in the catalysis of peptide bond formation with a pKa of 7.4 (1). Accordingly, with the crystal structure in hand (10), Steitz and co-workers proposed residue A2451 (Escherichia coli numbering is used throughout) to be the catalytic residue, since the N3 of the base lies within the hydrogen bonding distance of an analog designed to mimic the tetrahedral intermediate formed during transpeptidation (13). Serious biochemical and genetic reservations were raised concerning the acid–base catalytic mechanism proposed, not the least being that the pKa of the N3 of A2451 would have to be raised by at least 6 pH units to approach neutrality. Almost as rapidly as the proposal had been presented, sufficient evidence accumulated to suggests that this residue almost certainly did not participate in catalysis by the mechanism suggested (12,14,15).
Subsequently, the attention turned to identifying other residue(s) that might be responsible for the pH dependence of the reaction. Two favored candidate structures with near-neutral pKas were proposed (16), namely, the wobble pairs A2450-C2063 and A2453-C2499. An A-C wobble pair adopts the same geometry as a G-U wobble pair, but the pKa on the N1 of the adenosine is shifted toward neutrality, pH 6.2 (Figure 1A) (17). This perturbation is 2–3 pH units, but the local environment about the A-C wobble pair could further influence the acidity of this group. The conformational changes that had been shown by structure probing of the E.coli 50S subunit to be induced by alterations in both pH and monovalent cation concentration (12) now led to the consideration that pH-dependent conformational transitions might be physiologically relevant (18). Recently, this notion has been taken a step further (19), with the hypothesis that the relevant A-C wobble pair might act as a conformational switch. Whereas crystallographic evidence does support the requirement for conformational change during peptidyl transfer, there is no suggestion at all, however, that these changes involve either of these two wobble pairs (20).
Figure 1. Single and double mutations constructed at the A2450-C2063 and A2453-C2063 wobble base pairs. (A) Orientation of the A2450-C2063 and A2453-C2499 wobble base pairs in the peptidyltransferase center of the H.marismortui 50S subunit (10). A2451 is in red. (B) Single mutations are predicted to yield Watson–Crick geometries while destroying the near-neutral pKa found in A-C wobbles. Double mutations also do not contain a near-neutral pKa; however, these retain the wobble geometry. Hydrogen bonds resulting from the perturbed pKa at the N1 of adenosine in the context of the A-C wobble are in red.
Here, we report our structure probing results with single and double mutations at both of these wobble pairs. We conclude that the pair responsible for the pH sensitivity of the active site conformation is A2453-C2499 and argue that the structural changes induced are unlikely to be related to peptide bond formation.
MATERIALS AND METHODS
Site-directed mutagenesis
Mutagenesis was performed using QuikChange (Stratagene) on plasmids pLK35 and pLSH25. pLK35 contains the E.coli rrnB operon under control of the inducible phage PL promoter, while pLSH25 contains the rrnB operon containing the helix 25 streptavidin tag under the control of the phage PL promoter (21).
Polysome gradients
The sucrose gradient analysis of ribosome species from mutants grown to an OD600 of 0.6 was carried out as described previously (22). The gradient fractions containing 50S subunits, 70S ribosomes or polysomes were collected, and rRNA was extracted and analyzed by primer extension (23). Relative intensities of bands corresponding to extension products from the mutant and wild-type templates were determined using a Fuji BAS-2500 PhosphorImager.
Tagged ribosome preparation, purification and chemical modification
Plasmids containing mutations in the A-C wobble pairs were transformed into E.coli strain pop2136, which contains a temperature-sensitive cI repressor mutant, cI857. Mutant rRNA is expressed from the PL promoter by raising the temperature to 42°C. Preparation and affinity purification of mutant ribosomes were performed as described previously (21). Chemical probing was performed as described (12).
In vitro assays
In vitro transcription-translation assays were performed as described previously (15).
RESULTS
Construction of rRNA mutants
We sought to test the correlation of pH-dependent DMS reactivity of A2451 with the identity of the 23S rRNA A2450-C2063 or A2453-C2499 wobble base pairs by introducing both single and double mutations at each pair. We constructed G-U wobble pairs (A2450G-C2063U or A2453G-C2499U) in order to investigate the effect of removing the perturbed pKa in each A-C pair (Figure 1B). Single A to G mutations at A2450 or A2453 and single C to U mutations at C2063 or C2499 were also constructed. These single mutations are predicted to change their respective A-C wobble base pair into Watson–Crick A-U or G-C pairs (Figure 1B) and, as a result of these mutations, any perturbed, near-neutral pKa in the respective A-C wobble pair should be lost. Our aim is to limit structural changes introduced by mutation of these residues, but we cannot exclude the possibility that additional perturbations of the local structure were introduced.
Mutations were introduced into a plasmid-encoded E.coli rrnB operon under control of the inducible phage PL promoter. Essentially, the wild-type growth of E.coli was observed upon expression of plasmid-encoded ribosomes containing single or double mutations at either of the A-C wobble pairs, provided this was in a background of wild-type, chromosomally encoded ribosomes. However, none of the mutants constructed was capable of serving as the sole source of ribosomes in a strain of E.coli lacking its seven endogenous rRNA operons (24). We conclude that all the rRNA mutations constructed in this study result in recessive lethal phenotypes. This is in contrast to the report of the Strobel group (19) that their double mutant, A2450G-C2063U, possesses a dominant lethal phenotype, but which might well be a consequence of the additional S12 mutation (rpsL121) in the rRNA deletion strain (25) used in that work.
Mutant ribosomes enter polysomes in vivo
Our initial characterization of mutants was performed in an E.coli strain containing its full complement of seven, wild-type, chromosomal rrn operons. Quantitation of mutant versus wild-type rRNAs from 50S subunits, 70S ribosomes and polysome fractions indicated that mutant ribosomes were capable of entering translating pools (Table 1). Of all the mutants, either single or double, only the C2063U mutant was strongly inhibited from entering polysome pools despite assembling and associating into ribosomes (29% in polysomes versus 66% in 70S ribosomes). We noted that the formation of 50S or 70S ribosomes containing the A2450G mutation, either in isolation or in the context of the A2450G-C2063U double mutation, was compromised compared to that observed for the other mutants. Furthermore, 50S subunits with the A2453G-C2499U double mutation showed a reduced ability to associate with 30S subunits to form 70S ribosomes. The presence of all the mutants, at various levels, in polysomes, would therefore seem to indicate that they are capable of engaging in protein synthesis, although presumably not at the rate required for supporting cell viability.
Table 1. Percentage incorporation of mutant ribosomes into ribosome gradient fractions
In vitro translation by purified mutant ribosomes
To establish the functionality of the double mutants unequivocally, we chose to examine them in an in vitro coupled transcription-translation assay (15). First, to obtain highly purified populations of the mutant ribosomes, we introduced each of the double mutations into an rRNA expression plasmid containing a streptavidin-binding aptamer in helix 25 of 23S rRNA (21). Other tagging systems, using RNA inserts in 23S rRNA that bind MS2 coat protein (26) or eukaryotic splicing factor U1A (19), have also been developed more recently. The advantage of the Leonov (21) and Youngman (26) systems is that ribosomes carrying the tag are simply attached to an affinity matrix from which they are eluted without further manipulation. The Youngman system (26) has the additional advantage that the RNA tag is small (17 nt). The Hesslein system (19) not only incorporates a relatively large tag (100 nt) but also results in purified subunits that still have the U1A protein attached.
After growth and induction of expression of the mutant rRNA, the cells were lysed and samples isolated which contained both wild-type and mutant ribosomes. Purification of the mutant ribosomes was achieved by binding to a streptavidin resin. For each double mutant, a greater than 98% pure population of mutant ribosomes was achieved (Figure 2), with the tagged 50S subunits initially representing 30–50% of the total subunits, depending on the mutant.
Figure 2. A representative quantification of purified streptavidin-tagged ribosomes by strong stop primer extension. All 50S subunit preparations from all the mutants purified gave similar results. Lane 1: primer alone. Lane 2: primer extension on rRNA from chromosomally encoded ribosomes. Lane 3: primer extension on rRNA from both chromosome and plasmid (streptavidin-tagged) encoded ribosomes after streptavidin bead purification. The ratio of streptavidin-tagged to chromosomally encoded product in lane 3 is 98%, as determined by densitometry. Oligonucleotide used for strong stop primer extension: 5'-GAACTCTTGGGCGGTATCAGCC-3'.
The effect of the RNA tag in helix 25 on ribosome function was determined by assaying wild-type 50S subunits and purified, tagged 50S subunits carrying no other mutation, each with wild-type 30S subunits. The ribosomes were assayed in the coupled transcription-translation system for their ability to incorporate free methionine into protein using mRNA transcribed from a plasmid DNA template. No difference in activity was detected using several preparations of each subunit (data not shown), indicating that in this in vitro assay of complete protein synthesis, the RNA tag is inert. This result confirms and extends the observation that the placing of the streptavidin-binding aptamer in helix 25 has no effect on assembly (21). Next, purified 50S subunits containing either of the double mutations A2450G-C2063U or A2453G-C2499U, in association with wild-type 30S subunits, were assayed. The 50S subunits containing A2450G-C2063U or A2453G-C2499U double mutations synthesized proteins at 70 and 40% the rate of aptamer-tagged wild-type ribosomes, respectively (Figure 3). Thus, in agreement with the polysome gradient profiles, ribosomes carrying mutations at either of the A-C wobble pairs are capable of engaging in protein synthesis.
Figure 3. Incorporation of methionine into protein in a coupled transcription-translation system (15) by A-C wobble double mutant ribosomes. Closed squares: wild-type, tagged ribosomes; closed triangles: A2450G-C2063U mutant ribosomes; closed diamonds: A2453G-C2499U mutant ribosomes.
DMS reactivity of residue A2451 of mutant ribosomes
Early works showed that peptidyltransferase could undergo an active/inactive transition by manipulating monovalent cation concentrations and temperature (11). More recently, we have shown that ribosomes inactivated by monovalent cation depletion exhibit pH-dependent DMS reactivity at A2451 due to conformational change at this residue (12). We therefore wanted to test whether either of the A-C wobble pairs was responsible for the pH-dependent DMS reactivity of A2451 by chemical probing of streptavidin-purified mutant 50S subunits. First, however, we confirmed that the presence of the streptavidin-binding aptamer in helix 25 had no effect on the pH-dependent DMS reactivity of otherwise wild-type ribosomes (Figure 4A). The 50S subunits were inactivated by monovalent cation depletion and then assayed for pH-dependent DMS reactivity at A2451, after adding back 150 mM NH4+, with or without the heat incubation step critical for restoring the peptidyl transfer capacity of the subunits. The tagged ribosomes displayed pH-dependent DMS reactivity at A2451 prior to incubation at 37°C, in a manner indistinguishable from that of wild-type ribosomes with no aptamer tag (12). Again, therefore, the presence of the tag does not appear to interfere with the pH-dependent conformational change at residue A2451 seen upon inactivation/activation.
Figure 4. pH-dependent DMS reactivity of A2451 in A-C wobble mutants. All chemical probing was performed in the presence of 150 mM NH4+. The activation of ribosomes is denoted by the presence of the 37°C heating step. Unm: unmodified. (A) Ribosomes carrying only the helix 25 streptavidin-binding aptamer display pH-dependent DMS reactivity in inactive ribosomes at A2451. (B) Ribosomes carrying the A2450G mutation display a reversed DMS modification profile compared to wild-type, inactivated ribosomes. (C) Ribosomes carrying the A2450G-C2063U mutations display high, invariant DMS reactivity at A2451. No DMS reactivity is observed at A2451 in ribosomes carrying mutations A2453G (D), C2499U (E) or A2453G-C2499U (F).
Ribosomes containing the C2063U mutation, either alone (data not shown) or in the context of the A2450G-C2063U double mutation (Figure 4C), displayed high reactivity at A2451 at each pH examined, and whether or not the heat activation step had been carried out. A similar result with the double mutant was obtained by Hesslein et al. (19). Conversely, ribosomes carrying the A2450G mutation alone showed clear pH-dependent DMS reactivity at A2451 in both active and inactive ribosomes (Figure 4B). The degree of DMS reactivity at A2451 was, however, greater in inactive ribosomes, with the extent of modification at this base being reduced after incubation of the ribosomes at 37°C. Unlike wild-type E.coli ribosomes, where DMS reactivity at A2451 is highest at higher pH (pH 8.5), in both active and inactive A2450G mutant ribosomes (Figure 4A), the DMS reactivity was highest at lower pH (pH 6.5). These results indicate that, while the C2063U mutation perturbs the structure in this region sufficiently to cause high DMS reactivity at A2451 under all conditions examined, the A2450G mutation leaves the ionizable group intact, A2453, capable of causing conformational change at A2451 in response to pH.
Finally, we examined the consequences of mutating the A2453-C2499 wobble base pair. Ribosomes carrying the A2453G (Figure 4D) or C2499U (Figure 4E) mutations, individually or together (Figure 4F), lost their pH-dependent DMS reactivity at A2451 in both inactive and active ribosomes. Taken together, these results are consistent with the A2453-C2499 wobble base pair providing the perturbed, near-neutral pKa responsible for pH-dependent DMS reactivity at A2451.
DISCUSSION
We have shown by mutagenesis and chemical probing that the A2453-C2499 wobble base pair in 23S rRNA is essential for the pH-dependent structural rearrangement of A2451 in the peptidyltransferase center of the 50S subunit. The pH-dependent DMS reactivity at A2451 is lost with all three mutations examined for A2453-C2499. Given the highly specific nature of perturbed pKas in A-C wobble base pairs, this is precisely to be expected of mutations of an A-C wobble pair responsible for pH-dependent conformational change. In contrast, the A2450G mutant retained pH-dependent DMS reactivity at A2451, indicating that this base in the A2450-C2063 wobble pair is not the cause of the pH-dependent conformational change. The high level of DMS reactivity of A2451 observed when the C2063U mutation is present, either alone or in combination with A2450G, presumably indicates a structural change that leaves A2451 accessible to solvent.
While the A2450G mutant ribosomes retain pH-dependent DMS reactivity at A2451, the pattern of DMS reactivity at this base is reversed compared to wild type. That is, A2451 is more reactive to DMS modification at pH 6.5 than at 8.5 in A2450G mutant ribosomes. A similarly reversed pattern has also been observed in H.marismortui (27), as well as in monovalent cation-depleted ribosomes from Thermus thermophilus carrying the G2447A mutation (data not shown). Other investigations of pH-dependent changes in the peptidyltransferase center in other organisms have also met with mixed results. Thus, no pH-dependent changes at A2451 were observed in Thermus aquaticus or Mycobacterium smegmatis (18) or in T.thermophilus wild-type ribosomes (data not shown). However, the G2447U mutant of M.smegmatis showed a similar pH-dependent modification pattern to that of wild-type E.coli (18). It should, however, be noted that despite the high level of conservation of residues within the peptidyltransferase center of these organisms, there are differences in local stacking that will lead to different placements of ionizable groups. These subtle differences in interactions of specific residues may afford an explanation for the differences in viability of mutations of the same base in one organism compared with another. The simplest explanation for the contradictory behavior of A2451 in these species and mutants is that pH-dependent conformational change at the peptidyltransferase center is not a conserved feature of the ribosome.
It is reasonable to expect that any model for the catalysis of peptide bond formation should be applicable to all ribosomes, regardless of their source. The most highly (although not absolutely) conserved wobble pair, A2450-C2063, appears not to be essential for the pH-dependent DMS reactivity of A2451 . The wobble pair apparently carrying the near-neutral pKa required for the structural change, A2453-C2499, is only moderately conserved when rRNA sequences from all domains of life are examined (Table 2). Conservation among the bacteria, as for the A2450-C2063 wobble pair, is very high, but not absolute. In the archaea, the majority of sequences have an A at position 2499, and it is fortuitous that the A-C wobble geometry was found in the crystal structure of H.marismortui. It is perhaps not surprising, therefore, that viable mutants at A2453 in bacterial (data not shown) and archaeal species (28) and at C2499 in archaea (29) have been obtained. The A-C identity breaks down completely in the eucarya, where for all the sequences available, the residues are U-U. Thus, consideration of either of these two wobble pairs as candidates required for a universally conserved and essential conformational change during catalysis can no longer be entertained.
Table 2. Nucleotide frequency (%) of the A2453-C2499 and A2450-C2063 wobble base pairs
Over the years, the notion that peptidyl transfer might be a reaction that may not require direct involvement of components of the ribosome in transition state intermediates has been presented (30,31), but only very recently has this been tested directly. In a recent thermodynamic study, Sievers et al. (32) have shown that the role of the ribosome, at least in peptide bond formation, is simply to position the tRNAs in an appropriate fashion for the reaction to proceed. They argue that there is no requirement for a contribution to conventional catalysis by residues at the active site. If this is indeed the case, then the presence or absence of ionizable groups in the peptidyltransferase center has no bearing on peptide bond formation. The fact that in both the double mutants examined here, A2451 is either exposed to solvent (A2450G-C2063U; Figure 4C) or not (A2453G-C2499U; Figure 4F), but that both mutants are capable of peptide bond formation (Figure 3), suggests that there is no correlation between the accessibility of A2451 to solvent and peptidyltransferase capacity of the active site. Rather, the A-C wobble pairs of this study may function to maintain the correct structure of the active site in the context of the identity of other residues in the vicinity, not all of which are absolutely conserved among species.
ACKNOWLEDGEMENTS
We thank the members of the Dahlberg laboratory for helpful comments and particularly Steven Gregory for his suggestions and critical reading of the manuscript. We thank Petr Sergiev for the affinity tagged rDNA plasmid and Sunthorn Pond-Tor for excellent technical assistance. This research was supported by US National Institutes of Health grant GM19756 to A.E.D.
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* To whom correspondence should be addressed. Tel: +1 401 863 2223; Fax: +1 401 863 1182; Email: Albert_Dahlberg@Brown.edu
Present address: Mark A. Bayfield, NICHD, NIH, Bethesda, MD 02892, USA
ABSTRACT
Peptide bond formation, catalyzed by the ribosomal peptidyltransferase, has long been known to be sensitive to monovalent cation concentrations and pH. More recently, we and others have shown that residue A2451 in the peptidyltransferase center of the Escherichia coli 50S ribosomal subunit changes conformation in response to alterations in pH, depending on ionic conditions and temperature. Two wobble pairs, A2453-C2499 and A2450-C2063, have been proposed as potential candidates to convey pH-dependent flexibility to the peptidyltransferase center. Each is presumed to possess a near-neutral pKa, and both lie in proximity to A2451. We show through mutagenesis and chemical probing that the identity of the A2453-C2499 base pair, but not the A2450-C2063 base pair, is critical for the pH-dependent structural rearrangement of A2451. We conclude that, while the A2453-C2499 base pair may be important for maintaining the structure of the active site in the E.coli peptidyltransferase center, its lack of conservation makes it, and consequently its near-neutral pKa, unlikely to contribute to function during peptide bond formation.
INTRODUCTION
For four decades, the mechanism by which the ribosome catalyzes peptide bond formation has been sought. Early works focused on attempts to determine which ribosomal protein (or set of proteins) constituted the peptidyltransferase enzyme, in concert with the then understanding that all enzymes were proteins. Accordingly, typical properties of the enzyme were assessed, e.g. its dependence upon magnesium ions and its pH profile (1). Frustratingly, although a number of proteins clearly enhanced peptide bond formation (2), none in isolation was shown capable of catalysis (3). The discovery of catalytic RNA in the 1980s (4) altered the direction of research. Early insights (5,6) into the evolution of the translation machinery were re-evaluated and remarkable attempts were made to establish that RNA alone, specifically 23S RNA, was capable of catalytic function (7–9). Not until the solution of the crystal structure of the Haloarcula marismortui large ribosomal subunit was achieved, however, did it become absolutely clear that the peptidyltransferase center was completely devoid of protein (10). Since then, the attention has focused almost exclusively on attempts to determine which RNA residue(s) constitute the active site.
The investigation of the ionic requirements of the peptidyltransferase reaction revealed that the active site was conformationally labile. The removal of monovalent cations, in particular, resulted in ribosomes that were incapable of forming peptide bonds (as measured by the puromycin reaction). Inactivation was not irreversible, however; activity could be restored by adding back monovalent cations but a heating step was also required (11), presumably to help re-establish the native, active conformation. We (12) recapitulated these earlier findings and showed that the accessibility of residues in the active site and elsewhere in the ribosome to modification by dimethyl sulfate (DMS) was correlated with the active/inactive transition. At the same time, we demonstrated that there was a change in reactivity of certain residues to DMS that was dependent upon pH, and which also correlated with the active/inactive transition.
The sharp pH dependence of the peptidyltransferase reaction, regardless of whether the substrate for the puromycin reaction was full-length tRNA or the terminal CCA T1 ribonuclease fragment, suggested participation of a functional group in the catalysis of peptide bond formation with a pKa of 7.4 (1). Accordingly, with the crystal structure in hand (10), Steitz and co-workers proposed residue A2451 (Escherichia coli numbering is used throughout) to be the catalytic residue, since the N3 of the base lies within the hydrogen bonding distance of an analog designed to mimic the tetrahedral intermediate formed during transpeptidation (13). Serious biochemical and genetic reservations were raised concerning the acid–base catalytic mechanism proposed, not the least being that the pKa of the N3 of A2451 would have to be raised by at least 6 pH units to approach neutrality. Almost as rapidly as the proposal had been presented, sufficient evidence accumulated to suggests that this residue almost certainly did not participate in catalysis by the mechanism suggested (12,14,15).
Subsequently, the attention turned to identifying other residue(s) that might be responsible for the pH dependence of the reaction. Two favored candidate structures with near-neutral pKas were proposed (16), namely, the wobble pairs A2450-C2063 and A2453-C2499. An A-C wobble pair adopts the same geometry as a G-U wobble pair, but the pKa on the N1 of the adenosine is shifted toward neutrality, pH 6.2 (Figure 1A) (17). This perturbation is 2–3 pH units, but the local environment about the A-C wobble pair could further influence the acidity of this group. The conformational changes that had been shown by structure probing of the E.coli 50S subunit to be induced by alterations in both pH and monovalent cation concentration (12) now led to the consideration that pH-dependent conformational transitions might be physiologically relevant (18). Recently, this notion has been taken a step further (19), with the hypothesis that the relevant A-C wobble pair might act as a conformational switch. Whereas crystallographic evidence does support the requirement for conformational change during peptidyl transfer, there is no suggestion at all, however, that these changes involve either of these two wobble pairs (20).
Figure 1. Single and double mutations constructed at the A2450-C2063 and A2453-C2063 wobble base pairs. (A) Orientation of the A2450-C2063 and A2453-C2499 wobble base pairs in the peptidyltransferase center of the H.marismortui 50S subunit (10). A2451 is in red. (B) Single mutations are predicted to yield Watson–Crick geometries while destroying the near-neutral pKa found in A-C wobbles. Double mutations also do not contain a near-neutral pKa; however, these retain the wobble geometry. Hydrogen bonds resulting from the perturbed pKa at the N1 of adenosine in the context of the A-C wobble are in red.
Here, we report our structure probing results with single and double mutations at both of these wobble pairs. We conclude that the pair responsible for the pH sensitivity of the active site conformation is A2453-C2499 and argue that the structural changes induced are unlikely to be related to peptide bond formation.
MATERIALS AND METHODS
Site-directed mutagenesis
Mutagenesis was performed using QuikChange (Stratagene) on plasmids pLK35 and pLSH25. pLK35 contains the E.coli rrnB operon under control of the inducible phage PL promoter, while pLSH25 contains the rrnB operon containing the helix 25 streptavidin tag under the control of the phage PL promoter (21).
Polysome gradients
The sucrose gradient analysis of ribosome species from mutants grown to an OD600 of 0.6 was carried out as described previously (22). The gradient fractions containing 50S subunits, 70S ribosomes or polysomes were collected, and rRNA was extracted and analyzed by primer extension (23). Relative intensities of bands corresponding to extension products from the mutant and wild-type templates were determined using a Fuji BAS-2500 PhosphorImager.
Tagged ribosome preparation, purification and chemical modification
Plasmids containing mutations in the A-C wobble pairs were transformed into E.coli strain pop2136, which contains a temperature-sensitive cI repressor mutant, cI857. Mutant rRNA is expressed from the PL promoter by raising the temperature to 42°C. Preparation and affinity purification of mutant ribosomes were performed as described previously (21). Chemical probing was performed as described (12).
In vitro assays
In vitro transcription-translation assays were performed as described previously (15).
RESULTS
Construction of rRNA mutants
We sought to test the correlation of pH-dependent DMS reactivity of A2451 with the identity of the 23S rRNA A2450-C2063 or A2453-C2499 wobble base pairs by introducing both single and double mutations at each pair. We constructed G-U wobble pairs (A2450G-C2063U or A2453G-C2499U) in order to investigate the effect of removing the perturbed pKa in each A-C pair (Figure 1B). Single A to G mutations at A2450 or A2453 and single C to U mutations at C2063 or C2499 were also constructed. These single mutations are predicted to change their respective A-C wobble base pair into Watson–Crick A-U or G-C pairs (Figure 1B) and, as a result of these mutations, any perturbed, near-neutral pKa in the respective A-C wobble pair should be lost. Our aim is to limit structural changes introduced by mutation of these residues, but we cannot exclude the possibility that additional perturbations of the local structure were introduced.
Mutations were introduced into a plasmid-encoded E.coli rrnB operon under control of the inducible phage PL promoter. Essentially, the wild-type growth of E.coli was observed upon expression of plasmid-encoded ribosomes containing single or double mutations at either of the A-C wobble pairs, provided this was in a background of wild-type, chromosomally encoded ribosomes. However, none of the mutants constructed was capable of serving as the sole source of ribosomes in a strain of E.coli lacking its seven endogenous rRNA operons (24). We conclude that all the rRNA mutations constructed in this study result in recessive lethal phenotypes. This is in contrast to the report of the Strobel group (19) that their double mutant, A2450G-C2063U, possesses a dominant lethal phenotype, but which might well be a consequence of the additional S12 mutation (rpsL121) in the rRNA deletion strain (25) used in that work.
Mutant ribosomes enter polysomes in vivo
Our initial characterization of mutants was performed in an E.coli strain containing its full complement of seven, wild-type, chromosomal rrn operons. Quantitation of mutant versus wild-type rRNAs from 50S subunits, 70S ribosomes and polysome fractions indicated that mutant ribosomes were capable of entering translating pools (Table 1). Of all the mutants, either single or double, only the C2063U mutant was strongly inhibited from entering polysome pools despite assembling and associating into ribosomes (29% in polysomes versus 66% in 70S ribosomes). We noted that the formation of 50S or 70S ribosomes containing the A2450G mutation, either in isolation or in the context of the A2450G-C2063U double mutation, was compromised compared to that observed for the other mutants. Furthermore, 50S subunits with the A2453G-C2499U double mutation showed a reduced ability to associate with 30S subunits to form 70S ribosomes. The presence of all the mutants, at various levels, in polysomes, would therefore seem to indicate that they are capable of engaging in protein synthesis, although presumably not at the rate required for supporting cell viability.
Table 1. Percentage incorporation of mutant ribosomes into ribosome gradient fractions
In vitro translation by purified mutant ribosomes
To establish the functionality of the double mutants unequivocally, we chose to examine them in an in vitro coupled transcription-translation assay (15). First, to obtain highly purified populations of the mutant ribosomes, we introduced each of the double mutations into an rRNA expression plasmid containing a streptavidin-binding aptamer in helix 25 of 23S rRNA (21). Other tagging systems, using RNA inserts in 23S rRNA that bind MS2 coat protein (26) or eukaryotic splicing factor U1A (19), have also been developed more recently. The advantage of the Leonov (21) and Youngman (26) systems is that ribosomes carrying the tag are simply attached to an affinity matrix from which they are eluted without further manipulation. The Youngman system (26) has the additional advantage that the RNA tag is small (17 nt). The Hesslein system (19) not only incorporates a relatively large tag (100 nt) but also results in purified subunits that still have the U1A protein attached.
After growth and induction of expression of the mutant rRNA, the cells were lysed and samples isolated which contained both wild-type and mutant ribosomes. Purification of the mutant ribosomes was achieved by binding to a streptavidin resin. For each double mutant, a greater than 98% pure population of mutant ribosomes was achieved (Figure 2), with the tagged 50S subunits initially representing 30–50% of the total subunits, depending on the mutant.
Figure 2. A representative quantification of purified streptavidin-tagged ribosomes by strong stop primer extension. All 50S subunit preparations from all the mutants purified gave similar results. Lane 1: primer alone. Lane 2: primer extension on rRNA from chromosomally encoded ribosomes. Lane 3: primer extension on rRNA from both chromosome and plasmid (streptavidin-tagged) encoded ribosomes after streptavidin bead purification. The ratio of streptavidin-tagged to chromosomally encoded product in lane 3 is 98%, as determined by densitometry. Oligonucleotide used for strong stop primer extension: 5'-GAACTCTTGGGCGGTATCAGCC-3'.
The effect of the RNA tag in helix 25 on ribosome function was determined by assaying wild-type 50S subunits and purified, tagged 50S subunits carrying no other mutation, each with wild-type 30S subunits. The ribosomes were assayed in the coupled transcription-translation system for their ability to incorporate free methionine into protein using mRNA transcribed from a plasmid DNA template. No difference in activity was detected using several preparations of each subunit (data not shown), indicating that in this in vitro assay of complete protein synthesis, the RNA tag is inert. This result confirms and extends the observation that the placing of the streptavidin-binding aptamer in helix 25 has no effect on assembly (21). Next, purified 50S subunits containing either of the double mutations A2450G-C2063U or A2453G-C2499U, in association with wild-type 30S subunits, were assayed. The 50S subunits containing A2450G-C2063U or A2453G-C2499U double mutations synthesized proteins at 70 and 40% the rate of aptamer-tagged wild-type ribosomes, respectively (Figure 3). Thus, in agreement with the polysome gradient profiles, ribosomes carrying mutations at either of the A-C wobble pairs are capable of engaging in protein synthesis.
Figure 3. Incorporation of methionine into protein in a coupled transcription-translation system (15) by A-C wobble double mutant ribosomes. Closed squares: wild-type, tagged ribosomes; closed triangles: A2450G-C2063U mutant ribosomes; closed diamonds: A2453G-C2499U mutant ribosomes.
DMS reactivity of residue A2451 of mutant ribosomes
Early works showed that peptidyltransferase could undergo an active/inactive transition by manipulating monovalent cation concentrations and temperature (11). More recently, we have shown that ribosomes inactivated by monovalent cation depletion exhibit pH-dependent DMS reactivity at A2451 due to conformational change at this residue (12). We therefore wanted to test whether either of the A-C wobble pairs was responsible for the pH-dependent DMS reactivity of A2451 by chemical probing of streptavidin-purified mutant 50S subunits. First, however, we confirmed that the presence of the streptavidin-binding aptamer in helix 25 had no effect on the pH-dependent DMS reactivity of otherwise wild-type ribosomes (Figure 4A). The 50S subunits were inactivated by monovalent cation depletion and then assayed for pH-dependent DMS reactivity at A2451, after adding back 150 mM NH4+, with or without the heat incubation step critical for restoring the peptidyl transfer capacity of the subunits. The tagged ribosomes displayed pH-dependent DMS reactivity at A2451 prior to incubation at 37°C, in a manner indistinguishable from that of wild-type ribosomes with no aptamer tag (12). Again, therefore, the presence of the tag does not appear to interfere with the pH-dependent conformational change at residue A2451 seen upon inactivation/activation.
Figure 4. pH-dependent DMS reactivity of A2451 in A-C wobble mutants. All chemical probing was performed in the presence of 150 mM NH4+. The activation of ribosomes is denoted by the presence of the 37°C heating step. Unm: unmodified. (A) Ribosomes carrying only the helix 25 streptavidin-binding aptamer display pH-dependent DMS reactivity in inactive ribosomes at A2451. (B) Ribosomes carrying the A2450G mutation display a reversed DMS modification profile compared to wild-type, inactivated ribosomes. (C) Ribosomes carrying the A2450G-C2063U mutations display high, invariant DMS reactivity at A2451. No DMS reactivity is observed at A2451 in ribosomes carrying mutations A2453G (D), C2499U (E) or A2453G-C2499U (F).
Ribosomes containing the C2063U mutation, either alone (data not shown) or in the context of the A2450G-C2063U double mutation (Figure 4C), displayed high reactivity at A2451 at each pH examined, and whether or not the heat activation step had been carried out. A similar result with the double mutant was obtained by Hesslein et al. (19). Conversely, ribosomes carrying the A2450G mutation alone showed clear pH-dependent DMS reactivity at A2451 in both active and inactive ribosomes (Figure 4B). The degree of DMS reactivity at A2451 was, however, greater in inactive ribosomes, with the extent of modification at this base being reduced after incubation of the ribosomes at 37°C. Unlike wild-type E.coli ribosomes, where DMS reactivity at A2451 is highest at higher pH (pH 8.5), in both active and inactive A2450G mutant ribosomes (Figure 4A), the DMS reactivity was highest at lower pH (pH 6.5). These results indicate that, while the C2063U mutation perturbs the structure in this region sufficiently to cause high DMS reactivity at A2451 under all conditions examined, the A2450G mutation leaves the ionizable group intact, A2453, capable of causing conformational change at A2451 in response to pH.
Finally, we examined the consequences of mutating the A2453-C2499 wobble base pair. Ribosomes carrying the A2453G (Figure 4D) or C2499U (Figure 4E) mutations, individually or together (Figure 4F), lost their pH-dependent DMS reactivity at A2451 in both inactive and active ribosomes. Taken together, these results are consistent with the A2453-C2499 wobble base pair providing the perturbed, near-neutral pKa responsible for pH-dependent DMS reactivity at A2451.
DISCUSSION
We have shown by mutagenesis and chemical probing that the A2453-C2499 wobble base pair in 23S rRNA is essential for the pH-dependent structural rearrangement of A2451 in the peptidyltransferase center of the 50S subunit. The pH-dependent DMS reactivity at A2451 is lost with all three mutations examined for A2453-C2499. Given the highly specific nature of perturbed pKas in A-C wobble base pairs, this is precisely to be expected of mutations of an A-C wobble pair responsible for pH-dependent conformational change. In contrast, the A2450G mutant retained pH-dependent DMS reactivity at A2451, indicating that this base in the A2450-C2063 wobble pair is not the cause of the pH-dependent conformational change. The high level of DMS reactivity of A2451 observed when the C2063U mutation is present, either alone or in combination with A2450G, presumably indicates a structural change that leaves A2451 accessible to solvent.
While the A2450G mutant ribosomes retain pH-dependent DMS reactivity at A2451, the pattern of DMS reactivity at this base is reversed compared to wild type. That is, A2451 is more reactive to DMS modification at pH 6.5 than at 8.5 in A2450G mutant ribosomes. A similarly reversed pattern has also been observed in H.marismortui (27), as well as in monovalent cation-depleted ribosomes from Thermus thermophilus carrying the G2447A mutation (data not shown). Other investigations of pH-dependent changes in the peptidyltransferase center in other organisms have also met with mixed results. Thus, no pH-dependent changes at A2451 were observed in Thermus aquaticus or Mycobacterium smegmatis (18) or in T.thermophilus wild-type ribosomes (data not shown). However, the G2447U mutant of M.smegmatis showed a similar pH-dependent modification pattern to that of wild-type E.coli (18). It should, however, be noted that despite the high level of conservation of residues within the peptidyltransferase center of these organisms, there are differences in local stacking that will lead to different placements of ionizable groups. These subtle differences in interactions of specific residues may afford an explanation for the differences in viability of mutations of the same base in one organism compared with another. The simplest explanation for the contradictory behavior of A2451 in these species and mutants is that pH-dependent conformational change at the peptidyltransferase center is not a conserved feature of the ribosome.
It is reasonable to expect that any model for the catalysis of peptide bond formation should be applicable to all ribosomes, regardless of their source. The most highly (although not absolutely) conserved wobble pair, A2450-C2063, appears not to be essential for the pH-dependent DMS reactivity of A2451 . The wobble pair apparently carrying the near-neutral pKa required for the structural change, A2453-C2499, is only moderately conserved when rRNA sequences from all domains of life are examined (Table 2). Conservation among the bacteria, as for the A2450-C2063 wobble pair, is very high, but not absolute. In the archaea, the majority of sequences have an A at position 2499, and it is fortuitous that the A-C wobble geometry was found in the crystal structure of H.marismortui. It is perhaps not surprising, therefore, that viable mutants at A2453 in bacterial (data not shown) and archaeal species (28) and at C2499 in archaea (29) have been obtained. The A-C identity breaks down completely in the eucarya, where for all the sequences available, the residues are U-U. Thus, consideration of either of these two wobble pairs as candidates required for a universally conserved and essential conformational change during catalysis can no longer be entertained.
Table 2. Nucleotide frequency (%) of the A2453-C2499 and A2450-C2063 wobble base pairs
Over the years, the notion that peptidyl transfer might be a reaction that may not require direct involvement of components of the ribosome in transition state intermediates has been presented (30,31), but only very recently has this been tested directly. In a recent thermodynamic study, Sievers et al. (32) have shown that the role of the ribosome, at least in peptide bond formation, is simply to position the tRNAs in an appropriate fashion for the reaction to proceed. They argue that there is no requirement for a contribution to conventional catalysis by residues at the active site. If this is indeed the case, then the presence or absence of ionizable groups in the peptidyltransferase center has no bearing on peptide bond formation. The fact that in both the double mutants examined here, A2451 is either exposed to solvent (A2450G-C2063U; Figure 4C) or not (A2453G-C2499U; Figure 4F), but that both mutants are capable of peptide bond formation (Figure 3), suggests that there is no correlation between the accessibility of A2451 to solvent and peptidyltransferase capacity of the active site. Rather, the A-C wobble pairs of this study may function to maintain the correct structure of the active site in the context of the identity of other residues in the vicinity, not all of which are absolutely conserved among species.
ACKNOWLEDGEMENTS
We thank the members of the Dahlberg laboratory for helpful comments and particularly Steven Gregory for his suggestions and critical reading of the manuscript. We thank Petr Sergiev for the affinity tagged rDNA plasmid and Sunthorn Pond-Tor for excellent technical assistance. This research was supported by US National Institutes of Health grant GM19756 to A.E.D.
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