The UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5′- and 3′-ends of 16S rRNA as well as maturation of the 5′-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.
Ribosome maturation and assembly occur in a cooperative and ordered fashion (Noller and Nomura, 1987; Kaczanowska and Ryden-Aulin, 2007). In bacteria, a 50S ribosomal subunit associates with a 30S ribosomal subunit to form an active 70S ribosome. The 50S ribosomal subunit is comprised of 23S and 5S rRNAs and 33 ribosomal proteins, while the 30S ribosomal subunit is comprised of 16S rRNA and 21 ribosomal proteins (Wilson and Nierhaus, 2007). 16S, 23S and 5S rRNAs are cotranscribed as part of a large rRNA precursor. Before transcription is complete, ribosomal proteins associate with rRNA to form ribonucleoprotein complexes that are acted on by RNase III (Robertson et al., 1967). RNase III cleaves the initial transcript into 17S, 25S and 9S rRNA precursors that undergo further processing at their 5′- and 3′-termini to yield mature 16S, 23S and 5S rRNAs respectively (Gegenheimer et al., 1977; Nierhaus, 1991).
Our understanding of the steps by which rRNA precursors are processed to their mature forms is still incomplete. In Escherichia coli, RNase G and RNase E are required for the maturation of the 5′-terminus of 16S rRNA (Li et al., 1999a). RNase T is required for maturation of the 3′-terminus of 23S rRNA (Li et al., 1999b). RNase E is required for partial maturation of the 5′-terminus of 5S rRNA (Misra and Apirion, 1979), and RNase E and RNase T are required for the maturation of the 3′-terminus of 5S rRNA (Misra and Apirion, 1979; Li and Deutscher, 1995). RNases responsible for the final step(s) in maturation of the 3′-terminus of 16S rRNA, 5′-terminus of 23S rRNA and 5′-terminus of 5S rRNA remain unknown (Deutscher, 2009) (Fig. 1).
Reconstitution of active 30S and 50S ribosomal subunits has been performed in vitro using mature rRNAs and ribosomal proteins (Nomura and Erdmann, 1970; Wireman and Sypherd, 1974; Nierhaus, 1980; Green and Noller, 1999). However, the conditions required for in vitro reconstitution are far from physiological and it is well recognized that many additional proteins, which include processing, modification and assembly factors, are required for rRNA maturation and eventual 50S, 30S and 70S ribosome formation in vivo. Several of these factors, such as Era and ObgE, are essential for cell viability (Wilson and Nierhaus, 2007). While these accessory factors are clearly important, in many cases we lack a good understanding of their role in ribosome biogenesis (Bunner et al., 2010).
The UPF0054 protein family is highly conserved among prokaryotes. A gene encoding a member of this protein family is found in almost all sequenced bacterial genomes and it is one of the 206 genes that comprise the predicted minimal bacterial genome set (Gil et al., 2004). In some bacteria including Bacillus subtilis (Kobayashi et al., 2003), Haemophilus influenzae (Akerley et al., 2002) and Vibrio cholerae (B.W. Davies et al., in preparation), the respective homologue is essential. In the plant symbiont, Sinorhizobium meliloti, the respective homologue is not essential, but loss of its activity results in extreme pleiotropy and an inability of S. meliloti to form a symbiosis with plant hosts (Davies and Walker, 2008).
Here, we show that loss of the E. coli homologue, YbeY, results in viable cells with a pleiotropic phenotype and striking defects in ribosome function including decreased ribosome activity, reduced translational fidelity and altered translation initiation factor binding. Furthermore, we demonstrate that deletion of ybeY strongly affects the maturation of all three rRNAs causing a particularly strong defect in maturation of the 16S rRNA 5′- and 3′-termini, as well as maturation of the 5′-termini of 23S and 5S rRNAs. Strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase) additionally suggest a requirement for YbeY activity in rRNA maturation. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.
UPF0054 protein family members in bacteria are functionally equivalent
Disruption of the gene encoding the S. meliloti UPF0054 protein family member, SMc01113, results in extreme pleiotropy (Davies and Walker, 2008). Deletion of the E. coli UPF0054 family member gene, ybeY, from the reference strain MC4100 similarly results in an extremely pleiotropic phenotype that includes a modest decrease in growth rate in rich medium (Fig. 2A), but a significant sensitivity to numerous physiologically diverse stresses including β-lactam antibiotics, temperature, detergents and oxidative stress (Fig. 2B and C; Fig. S1). Deletion of ybeY from a second reference E. coli strain, MG1655, resulted in the same phenotypes (data not shown), indicating that the effects are not strain-specific. All ΔybeY mutant phenotypes were rescued by ectopic expression of ybeY (Fig. 2A–C; Fig. S1). Expression of either the S. meliloti homologue SMc01113 or the B. subtilis homologue yqfG rescues the ΔybeY mutant phenotypes as effectively as ybeY itself (Fig. 2A–C; Fig. S1). Conversely, expression of ybeY in the S. meliloti SMc01113 mutant background rescues all free-living and symbiotic phenotypes of the SMc01113 mutant (Fig. S2A–C and data not shown) (Jones et al., 2007; Davies and Walker, 2008). Taken together, these results demonstrate a universally conserved function for UPF0054 members in bacteria.
Deletion of ybeY causes defects in polysome formation, ribosome activity, translational fidelity and translation initiation factor binding
Compared with the polysome profile of the parental strain, the ΔybeY mutant has a decrease in polysomes and a large increase in both free 50S and free 30S ribosomal subunits relative to 70S ribosomes (Fig. 2D). Ectopic expression of ybeY in the ΔybeY mutant restored the normal polysome profile (Fig. S3A–C). After we had completed much of this work, a study investigating the role of YbeY in the E. coli heat shock response also reported that deletion of ybeY caused loss of polysomes (Rasouly et al., 2009); however, there was no mention of a potential function for YbeY in ribosome maturation.
The decreased polysome population led us to examine the functional state of the intact ribosomes in the ΔybeY mutant. We reconstituted an in vitro translation system with an S100 extract from the wild-type strain MC4100 and 70S ribosomes from either the ΔybeY mutant or its parent MC4100. Under saturating substrate conditions, ribosomes from the ΔybeY mutant had reduced translational activity compared with 70S ribosomes from MC4100 (Fig. 2E). Furthermore, both frameshifting and readthrough of nonsense codons were increased in vivo in the ΔybeY mutant, with the largest effect being for + 1 frameshifts (Fig. 2F and G). The accumulation of defects in ribosomal function and polysome assembly, could account for the pleiotropic nature of the ΔybeY mutant as many stress response programmes rely on the upregulation of defence proteins to ward off stress or to repair damage (Imlay and Linn, 1987; Friedberg et al., 2005).
The increased pools of free 30S and 50S ribosomal subunits found in the ΔybeY mutant suggest that a portion of these ribosomes may be defective in assembly of the 70S ribosome. In bacteria, translation initiation requires three protein initiation factors; IF1, IF2 and IF3. IF2 correctly positions the initiator tRNA in the ribosomal P site and promotes association of 30S and 50S ribosomal subunits. While IF1 is also known to stabilize the binding of the initiator tRNA, IF3 acts as a proofreading and anti-association factor (Kaczanowska and Ryden-Aulin, 2007). Analysis of total cell lysates showed only a slight but reproducible decrease in IF2 and an increase in IF3 in the ΔybeY mutant (Fig. 2H). However, we found a substantial decrease in IF2 present in the fraction containing 30S subunits isolated from the ΔybeY mutant, suggesting that many of the mutant 30S ribosomal subunits are not available for 70S ribosome assembly. We also observed a slight increase in IF3 associated with ΔybeY mutant 30S subunits and 70S ribosomes, but the change was modest compared with IF2. This suggests that the 30S ribosomal subunits in a ΔybeY mutant have a defect that prevents ribosome assembly and/or promotes disassembly of 70S ribosomes when assembly is attempted.
The ΔybeY mutant is defective in rRNA maturation
The ribosome is primarily composed of rRNA (Wilson and Nierhaus, 2007) and incomplete processing of rRNA can lead to defects in ribosome function (Wireman and Sypherd, 1974). Comparison of total rRNA profiles by agarose gel electrophoresis from MC4100 and the ΔybeY mutant showed the presence of a substantial amount of 17S rRNA in the ΔybeY mutant, as well as a faster migrating species, which we annotate as 16S*, indicating a defect in 16S rRNA maturation (Fig. 3A). 17S rRNA is the precursor of 16S rRNA (Fig. 1) and is generated from the initial rRNA transcript by RNase III cleavage (Gegenheimer et al., 1977). The aberrant 16S* rRNA species may be a product of misprocessing and/or degradation of the 17S or 16S rRNA. Ectopic expression of ybeY in the ΔybeY mutant restored normal 16S rRNA maturation (Fig. S3D). We were unable to assess the state of 5S and 23S rRNA processing in these total rRNA profile experiments (see below, however) because the difference between mature and immature 5S and 23S rRNAs is only a few nucleotides, respectively, and thus does not cause a detectable shift on agarose gels.
In strain MC4100, 30S ribosomal subunits contained mostly 16S rRNA along with some 17S rRNA, whereas 70S ribosomes contained only 16S rRNA (Fig. 3A). In contrast, 30S ribosomal subunits from the ΔybeY mutant contained much more 17S rRNA than 16S rRNA as well as a substantial amount of 16S* rRNA (Fig. 3A). Furthermore, there was also a substantial amount of 17S rRNA in 70S ribosomes isolated from the ΔybeY mutant (Fig. 3A). The accumulation of 17S rRNA was not due to the slower growth rate of the ΔybeY mutant. When the strains were grown in minimal medium where MC4100 and the ΔybeY mutant show similar doubling times, we still observed similar levels of 17S rRNA accumulation (data not shown). 17S rRNA is thought not to be competent for translation in vitro (Wireman and Sypherd, 1974). The increased abundance of 17S and 16S* rRNA in the 30S ribosomal subunits could account for altered translation initiation factor binding and the other translational defects described above for the ΔybeY mutant. Similarly, the increased abundance of 17S rRNA in 70S ribosomes could explain, at least in part, the reduced translation activity and decreased translational accuracy of the ΔybeY 70S ribosomes. Interestingly, 16S* rRNA was not found in ΔybeY 70S ribosomes suggesting that it is not competent for assembly into the 70S ribosome and may thus be a non-functional product of rRNA processing in the ΔybeY mutant.
In agreement with the observed increase in 17S rRNA in the ΔybeY mutant, Northern blot analysis showed that both 5′- and 3′-termini of 17S rRNA were present at much higher levels in total RNA extracted from the ΔybeY mutant than in total RNA from the parental strain MC4100 (Fig. 3B). Use of primer extension to map the 5′-terminus (Fig. 3C) and site-specific RNase H cleavage followed by Northern hybridization (Li et al., 1999b) to map the 3′-terminus of 16S rRNA (Fig. 3D) showed that the termini of the mature and immature 16S rRNA in total RNA extracted from the ΔybeY mutant and the parental strain MC4100 were identical. However, consistent with the above results, both immature 5′- and 3′-termini of 16S rRNA were present at higher levels in total RNA extracted from the ΔybeY mutant (Fig. 3C and D). We did not observe either 5′- or 3′-termini shorter than that of mature 16S rRNA, although, if the 5′- or 3′-terminus of the 16S* species were extensively degraded, the assays used would not have detected them.
Primer extension and site-specific RNase H cleavage were also used to determine the maturation state of 23S and 5S rRNAs. Strikingly, along with 16S rRNA, the maturation of the 5′- and 3′-termini of both 23S and 5S rRNA were also affected, with all four termini showing increased amounts of the immature form in the ΔybeY mutant (Fig. 3C and D), indicating that ybeY function is required for normal 5′ and 3′ processing of 16S, 23S and 5S rRNAs. Nevertheless, the most significant defects in the ΔybeY mutant appeared to be in the complete maturation of the 5′- and 3′-termini of 16S rRNA.
Rasouly et al. demonstrated a defect in the polysome profile of a ΔybeY mutant after heat shock (Rasouly et al., 2009). After shifting the cultures to 45°C for 1 h, we observed little mature 16S rRNA in the ΔybeY mutant by agarose gel electrophoresis; instead we saw two bands that migrate near the 17S and 16S* rRNA positions (Fig. S3E). In contrast, there were no significant changes in the rRNA profile of MC4100 samples shifted to 45°C compared with those at 37°C. As 17S rRNA is not competent for translation (Wireman and Sypherd, 1974), and 16S* rRNA appears to be excluded from 70S ribosomes (Fig. 3A), the increase in 17S and 16S* rRNA-like species and the decrease in 16S rRNA in the ΔybeY mutant following heat shock could explain the defect in polysome formation at elevated temperatures (Rasouly et al., 2009) and the overall temperature sensitivity of the ΔybeY mutant described above (Fig. 2C).
ybeY shows strong genetic interactions with rnc (RNase III), rnr (RNase R) and pnp (PNPase)
The serious defects in translation and rRNA maturation in the ΔybeY mutant suggested that YbeY might be involved in rRNA metabolism. To explore the cellular role of YbeY, we compared the rRNA profile of the ΔybeY mutant with that of several well-characterized E. coli RNase mutants disrupted in the following genes: cafA (RNase G), rnc (RNase III), rnr (RNase R), pnp (PNPase), rnd (RNase D), rnt (RNase T) and rph (RNase PH) (Fig. 4A). We also transduced each of these RNase mutations into the ΔybeY mutant background and examined the rRNA profiles of these double mutants (Fig. 4B). We then mapped the 5′- and 3′-termini of 16S, 23S and 5S rRNAs for each strain using primer extension and site-specific RNase H cleavage assays (Fig. 4C–H) as described above.
Profiles of total rRNA showed that, of all the mutants tested, the ΔybeY mutant had the greatest defect in maturation of 17S rRNA to 16S rRNA (Fig. 4A). Primer extension and site-specific RNase H cleavage assays confirmed this result showing high levels of both immature 5′ and 3′ 16S rRNA termini in the ΔybeY mutant compared with the other RNase mutants (Fig. 4C and D). As previously described (Li et al., 1999a), the ΔcafA mutant also showed severe defects in maturation of the 16S rRNA 5′-terminus. However, in contrast to the ΔybeY mutant, which accumulates the full 17S rRNA 5′ precursor (+ 115 nucleotides), the ΔcafA mutant accumulates the shorter + 66 nucleotides 5′ precursor (Fig. 4C). Also, of all the mutants tested, only the ΔybeY mutant showed accumulation of high levels of immature 16S rRNA 3′-terminus (Fig. 4D).
The introduction of either the rnc or rnr mutation into the ΔybeY mutant resulted in major alterations in the gross rRNA profile of the ΔybeY mutant (Fig. 4B). Strikingly, 17S rRNA was no longer present in the ΔybeY Δrnc mutant, instead 16S* rRNA and a new rRNA species migrating more slowly than 17S rRNA accumulated. Primer extension and site-specific RNase H cleavage assays confirmed this new precursor, which we designate 18S* rRNA, as a 16S rRNA derivative with additional nucleotides on both the 5′- and 3′-termini of 17S rRNA (Fig. 4C and D). Gegenheimer et al. reported a transient 18S rRNA species in a Δrnc mutant (Gegenheimer et al., 1977) that is matured to 16S rRNA and has a similar mobility as the new precursor species we observe. We have not mapped the termini of 18S* rRNA or the 18S species found by Gegenheimer et al. to the nucleotide level, but based on their relative mobility, we suggest that the 18S* rRNA observed in the ΔybeY Δrnc mutant may be 18S rRNA. If 18S* and 18S rRNA are identical, our data suggest that the absence of YbeY influences the processing of 18S rRNA to 16S rRNA in a Δrnc mutant. Further characterization of the 18S* rRNA species in the ΔybeY Δrnc mutant could be of much interest.
The ΔybeY Δrnr mutant showed a substantial decrease in 16S rRNA and reciprocal increase in both 17S and 16S* rRNA (Fig. 4B). This result was confirmed by mapping of the 5′- and 3′-termini (Fig. 4C and D). Furthermore, primer extension of 16S rRNA 5′-termini showed an increase in a set of new rRNA species of minor intensity that migrated between 17S and 16S rRNAs. These new species may represent partially mature or non-functional 16S-like rRNA species. RNase R has been shown to function as a scavenging RNase that removes non-functional rRNA from the cell (Cheng and Deutscher, 2003). Loss of this function is consistent with the appearance of 16S rRNA intermediates observed by primer extension in the ΔybeY Δrnr mutant. It also suggests that, in the absence of ybeY, the cell misprocesses large amounts of 17S rRNA generating abnormal species that RNase R normally helps to degrade.
Strikingly, we observed very little of the mature 16S rRNA 3′-terminus in either the ΔybeY Δrnr mutant or the ΔybeY Δpnp mutant rRNA (Fig. 4D). Deletion of either rnr or pnp alone does not significantly affect the maturation of the 16S rRNA 3′-terminus (Fig. 4D). The RNase(s) required for maturation of the 16S rRNA 3′-terminus has not been identified (Deutscher, 2009). Our results suggest that PNPase and RNase R may act in maturation of the 16S rRNA 3′-terminus and their role may be modulated by YbeY. Interestingly, while both the ΔybeY Δrnr mutant and the ΔybeY Δpnp mutant affect maturation of the 16S rRNA 3′-terminus, only the ΔybeY Δrnr mutant exhibits additional defects in 16S rRNA metabolism as shown by the appearance of extra aberrant rRNA species (Fig. 4B and C). This suggests that RNase R and YbeY interact in aspects of rRNA metabolism beyond 16S rRNA 3′-terminus maturation, such as quality control of rRNA.
Mapping of the 5′- and 3′-termini showed that deletion of ybeY also affected 5′ maturation of 23S and 5S rRNAs (Fig. 4E and H). The ΔybeY mutant had a greater effect in 23S and 5S rRNA maturation than nearly all of the RNase mutants tested. As expected, deletion of rnc abolishes formation of mature 23S rRNA 5′- and 3′-termini (King et al., 1984). Also as expected, deletion of rnt greatly inhibits maturation of the 23S and 5S rRNA 3′-termini (Li et al., 1999b). Interestingly, we have also found that in the ΔybeY Δrnt mutant the 17S rRNA precursor was extended by one or two nucleotides at the 3′-terminus (Fig. 4D), suggesting the possible involvement of RNase T in trimming the 17S precursor at the 3′-terminus prior to complete 3′ maturation of 16S rRNA. Deletion of pnp and rph also caused accumulation of 5′-immature 5S rRNA precursor implicating a role of these two RNases in this step of rRNA maturation (Fig. 4G).
RNase E is known to function in the maturation of the 5′-terminus of 16S rRNA (Li et al., 1999a), and the 5′- and 3′-termini of 5S rRNA (Misra and Apirion, 1979; Li and Deutscher, 1995). RNase E is essential in E. coli and temperature-sensitive mutants have been used to explore its potential roles in rRNA maturation (Li et al., 1999a). Using the rne-1 temperature-sensitive RNase E allele, we constructed an rne-1 ΔybeY::catR double mutant and attempted to explore their genetic interaction in rRNA processing at the non-permissive temperature (45°C). The rRNA profile is shown (Fig. S3F); mapping of the 16S rRNA termini showed that RNase E contributes to the maturation of the 5′-terminus of 16S rRNA as expected (Li et al., 1999a), but does not contribute significantly to the maturation of the 3′-terminus under the conditions used (data not shown). Unfortunately, it is difficult to interpret the results from the rne-1 ΔybeY::catR double mutant grown at the non-permissive temperature because of the strong temperature sensitivity of the ΔybeY mutant by itself (Rasouly et al., 2009; see also Fig. 2C and Fig. S3E). Because of this, effects of the rne-1 mutation at 45°C are overshadowed by effects of the ΔybeY deletion. A temperature-independent approach for conditional rne inactivation will be necessary for such experiments.
Growth rate analysis of the ΔybeY double mutants
We next examined the growth behaviour in rich medium of single and double mutants described above. Further strengthening a connection between YbeY and RNase R, we observed the greatest effect in the ΔybeY Δrnr mutant (Fig. 5). This mutant shows reduced growth rate in addition to an extended lag phase. Interestingly, while both the ΔybeY Δrnr and the ΔybeY Δpnp mutants affect 16S rRNA 3′-terminus maturation strongly, the ΔybeY Δpnp grows identically to the ΔybeY mutant, showing only a modest growth rate reduction. The change in growth behaviour of the ΔybeY Δrnr mutant compared with the ΔybeY mutant could be due to defects other than the incomplete maturation of the 16S rRNA, e.g. quality control of rRNA. Also, the significant accumulation of 16S* rRNA, a species that can be assembled in 30S ribosomal subunits, but not in functional 70S ribosomes (Fig. 3A) may be detrimental to the cell and be responsible for the growth behaviour of the ΔybeY Δrnr mutant.
We also observed a decrease in growth rate in the ΔybeY Δrnc mutant in rich medium (Fig. 5) compared with the respective single mutants, although this was less pronounced than for the ΔybeY Δrnr mutant. Thus, the strong genetic interaction observed in the ΔybeY Δrnr and ΔybeY Δrnc mutants is manifested at both the rRNA processing and physiological levels.
Highly conserved amino acid residues required for YbeY activity in vivo
All UPF0054 family members contain a conserved H3XH5XH motif (Fig. 6A). The presence of this motif has led to the categorization of UPF0054 family members as putative metal-dependent hydrolases and, more specifically, by some as putative metal-dependent proteases due to the presence of a similar motif found in certain mammalian proteases (E.V. Koonin, pers. comm.; Tatusov et al., 2001; Verma et al., 2002). The recently determined structures of E. coli YbeY and two additional UPF0054 family members (Oganesyan et al., 2003; Yeh et al., 2005; Zhan et al., 2005) support a putative metal-dependent hydrolytic function; however, no substrate has been identified for this protein family despite extensive screening (Oganesyan et al., 2003).
To study the structure–function relationship of the conserved UPF0054 motif and other highly conserved amino acids in this protein family, we mutated several conserved amino acid residues in ybeY on our complementation plasmid to Ala (Fig. 6A). We then determined the ability of these mutant ybeY alleles to complement the growth and rRNA processing defects of the ΔybeY mutant (Fig. 6B and C). The first His residue (H114) in the conserved H3XH5XH motif was required for partial recovery of growth of the ΔybeY mutant at 45°C and to rescue rRNA processing defects of the ΔybeY mutant, supporting the importance of this motif for YbeY function (Fig. 6B and C). Interestingly, changing the second and third His residues from this motif (H118, H124) to Ala did not have much of an effect on complementation. A highly conserved Arg residue (R59) was also found to be necessary for growth complementation. The loss of complementation by H114A and R59A alleles was not due to insufficient protein expression as plasmid-driven expression of all ybeY mutant alleles exceeded ybeY expression from the endogenous locus (Fig. S3G). It should be noted that some of the ybeY mutant alleles that did not show an effect on complementation when expressed from a multicopy plasmid, may do so if they are expressed at levels of YbeY normally present in wild-type cells.
We have shown that the functionally conserved UPF0054 protein family member, YbeY, influences rRNA maturation and ribosome formation in E. coli. Deletion of ybeY from E. coli results in defects in ribosome assembly and activity as well as in attenuation of 16S, 23S and 5S rRNA processing.
rRNA processing, ribosome maturation and translation are intertwined. Several reports have implicated partial ribosome assembly as a requirement for full rRNA maturation (Mangiarotti et al., 1974; 1975; Srivastava and Schlessinger, 1988) and, while deletion of specific RNases can lead to specific defects in rRNA maturation (Li and Deutscher, 1995; Li et al., 1999a,b), loss of non-RNase ribosome maturation factors can also lead to rRNA maturation defects. For example, both Era and ObgE are GTPases required for ribosome assembly. Era and ObgE are essential for viability in E. coli and loss of either activity causes accumulation of 17S rRNA (Inoue et al., 2003; Sato et al., 2005). Similarly, deletion of ribosome maturation factors RimB and RimP results in slower growth rates, reduced number of polysomes and accumulation of 16S rRNA precursor (Lövgren et al., 2004). Furthermore, loss of RsgA, a GTPase activated by the 30S ribosomal subunit, results in the accumulation of both 17S rRNA and another species shorter than 16S rRNA (Himeno et al., 2004). Our results could also mean that loss of YbeY affects a critical step in ribosome biogenesis and thereby slows rRNA maturation. It will be interesting to explore physical interactions of YbeY with the ribosome and rRNA directly as well as possible effects of YbeY on ribosomal proteins, e.g. protein modifications. Supporting a potential association of YbeY with the ribosome, recent work has identified interactions between YbeY and both 30S and 50S ribosomal proteins in a large-scale pull-down experiment (Arifuzzaman et al., 2006).
Active translation has also been suggested to be required for certain steps in rRNA maturation (Hayes and Vasseur, 1976; Sirdeshmukh and Schlessinger, 1985; Srivastava and Schlessinger, 1988). The dramatic decrease in IF2 binding to the 30S ribosomal subunit in the ΔybeY mutant (Fig. 2H) could suggest that YbeY is needed, directly or indirectly, to form the translation initiation complex. A decrease in efficiency of initiation complex formation could slow translation to a point that negatively impacts maturation of rRNA.
The strongest evidence of a role for YbeY in rRNA and/or ribosome maturation comes from the analysis of rRNA in strains carrying the ΔybeY mutation alone or in combination with other mutations in known RNases (Figs 3 and 4). RNases required for the processing of the 16S rRNA 3′-terminus, 23S rRNA 5′-terminus and 5S rRNA 5′-terminus have not been identified. Interestingly, we observe significant defects in processing of all of these termini in the ΔybeY mutant (Fig. 3C and D). The ΔybeY mutant shows a much stronger overall effect on rRNA maturation than any of the RNases tested, even those with a known role in rRNA processing (Fig. 4C–H). Only RNase III, which is required for the initial cleavages of the rRNA precursor, also affects the maturation of multiple rRNA termini. Deletion of both ybeY and rnc stabilizes the accumulation of an 18S-like rRNA precursor with similar mobility to that of a transient 18S rRNA species previously observed in an rnc mutant (Gegenheimer et al., 1977). Loss of YbeY activity may therefore exacerbate a specific rRNA maturation defect that is only observed in the absence of the RNase III required for the initial cleavage events in rRNA maturation.
Deletion of ybeY along with either rnr or pnp prevents, almost completely, the maturation of the 16S rRNA 3′-terminus (Fig. 4D), while maturation of all other rRNA termini proceeds to some degree in both of these mutants. Furthermore, large amounts of 16S rRNA are improperly processed in the ΔybeY Δrnr double mutant, as shown by the accumulation of additional aberrant 16S rRNA maturation intermediates. These results suggest that YbeY may function along with RNase R and PNPase in the maturation of the 16S rRNA 3′-terminus and potentially in other steps of rRNA metabolism such as rRNA quality control.
Site-directed mutagenesis of YbeY supports the requirement for one of the His residues (H114) in the highly conserved putative hydrolase domain and a highly conserved Arg residue (R59) for YbeY function in vivo (Fig. 6). For example, ectopic expression from a plasmid of ybeY mutants H114A and R59A in the ΔybeY mutant failed to complement the growth defect of the mutant strain at elevated temperatures. The H3XH5XH motif is postulated to co-ordinate a Zn2+ ion that could act catalytically in a hydrolysis reaction (Zhan et al., 2005). The crystal structure of YbeY also shows the highly conserved R59 pointing inward towards the H3XH5XH motif (Zhan et al., 2005). The location of R59 along with its positive charge could imply that R59 helps position a substrate near the H3XH5XH motif. Given the strong genetic interactions of ybeY with rnc, rnr and pnp (Fig. 4C–H) established here, along with the requirement of the putative hydrolase domain for ybeY activity, YbeY may also have RNase activity in the cell.
It is currently unclear why only one (H114) out of three His residues within the highly conserved H3XH5XH motif appears to be required for ybeY activity. It is possible that contributions of the other two His residues (H118, H124) towards YbeY activity may also be seen, when YbeY mutants are expressed at wild type-like levels. It is interesting to note the H114A and R59A mutants show similar sensitivity to growth at elevated temperature, but the rRNA processing defects are markedly different (Fig. 6B and C). While the rRNA profile of the H114A mutant resembles that of the ybeY mutant, the rRNA profile of the R59A mutant more closely resembles that of the MC4100, suggesting that the different mutations affect different functions of YbeY or affect the same rRNA processing function to different degrees.
Finally, although deletion of ybeY results in severe rRNA maturation defects suggesting a role in rRNA and/or ribosome maturation it remains possible that YbeY actually has an alternative function in the cell that impacts these processes. Although this remains a formal possibility, the simplest explanation for the striking effects of deletion of ybeY on rRNA processing and ribosome assembly is that YbeY functions directly in rRNA and/or ribosome maturation and assembly.
Strains, plasmids, growth conditions and DNA manipulations
Strains and plasmids are shown in Table S1. Strains were grown aerobically in Luria–Bertani (LB) medium at 37°C except for heat shock experiments where strains were grown at 45°C. Antibiotics for strain selection were used at the following concentrations, ampicillin 100 µg ml−1, chloramphenicol 20 µg ml−1 and kanamycin 25 µg ml−1. Deletion of ybeY and the addition of a C-terminal FLAG tag to the genomic copy of ybeY (strain BWD55) was performed using the methods of Wanner (Datsenko and Wanner, 2000). For deletion, ybeY was first replaced with a catR cassette creating the ΔybeY::catR mutant. The catR cassette was then excised to create a clean ΔybeY deletion mutant in MC4100. Deletion of ybeY maintains the ATG start of ybeY and the overlapping stop codon of the upstream gene ybeZ. Translation from the ATG start of ybeY terminates eight amino acids into the flip recombinase site of the λ-red gene targeting recombinase system (Datsenko and Wanner, 2000). DNA primers used for deletion of ybeY and addition of the C-terminal FLAG tag are listed in Table S2. Allele transfers were done by P1 transduction. DNA manipulations were performed according to the methods of Sambrook (Sambrook and Russell, 2001). Site-directed mutagenesis was performed using the Quikchange II kit from Stratagene. DNA primers used for site-directed mutagenesis are listed in Table S2. For complementation studies, ybeY, SMc01113 and yqfG were cloned downstream of the tetracycline promoter in pBR322 or the tryptophan promoter in pMS03. The DNA primers used for complementation cloning are shown in Table S2.
rRNA and polysomes were extracted from cells growing exponentially at 37°C, unless otherwise stated. Cells were pelleted, resuspended in buffer A (20 mM HEPES pH 7.5, 5 mM β-mercaptoethanol, 10 mM MgCl2, 50 mM NH4Cl and 0.1 mM PMSF) and lysed by repeated freeze/thaw cycles. After clarification by centrifugation, lysates were loaded on a 5–20% sucrose gradient. Polysomes and ribosomes were separated by ultracentrifugation followed by fractionation using a peristaltic pump. The A260 value was determined for each fraction.
rRNA was extracted from logarithmically growing cultures or separated ribosome fractions using Qiagen RNeasy Mini Kit. For gross analysis of 16S and 23S rRNA, 800 ng of rRNA was separated by synergel/agarose gel electrophoresis as described (Wachi et al., 1999) using 0.9% synergel and 0.7% agarose.
Primer extension assays were performed using Superscript II reverse transcriptase (Invitrogen) as per manufacturer's instructions with primers annealing to the 5′-terminal region of mature 16S, 23S and 5S rRNA respectively. The site-specific RNase H cleavage assay was performed as described (Li and Deutscher, 1995; Li et al., 1999a,b) followed by Northern hybridization using probes specific for the mature 3′-termini of 16S, 23S and 5S rRNAs respectively. One hundred nanograms of total RNA was used for each assay. Reaction products were separated on denaturing polyacrylamide gels; 10% polyacrylamide/7 M urea for primer extensions and 6% polyacrylamide/7 M urea for site-specific RNase H cleavage assays. DNA oligonucleotides for primer extension and RNase H mapping are shown in Table S2.
For the Northern blot in Fig. 2B, 250 ng of total MC4100 and ΔybeY RNA was separated on a 1% agarose gel containing 2% formamide. Probes used to identify 17S rRNA 5′- and 3′-termini are shown in Table S2. The probes were detected with the AlkPhos Direct Labeling Kit (Amersham).
For temperature shift assays, cultures were grown to exponential phase at 37°C. Samples were taken for rRNA analysis and the cultures were then shifted to 45°C. After 1 h samples were taken for rRNA analysis.
In vitro translation and lacZ assays
In vitro translation assays were performed essentially as described (Matthaei and Nirenberg, 1961). Exponentially growing cells were pelleted, resuspended in buffer A and lysed by French press. Lysates were centrifuged twice at 30 000 g and once at 100 000 g. The resulting supernatant was kept as the S100 fraction, and the pelleted ribosomes were washed and resuspended in buffer A. Ribosomes were further purified by pelleting at 200 000 g through a 20% sucrose cushion. Pelleted ribosomes were resuspended in buffer A and quantified by UV absorbance at 260 nm, and stored at − 80°C.
Mix I (25 µg of polyU mRNA and 10 pmoles of ribosomes) was pre-incubated at 37°C for 15 min in polyU-mix (20 mM HEPES pH 7.5, 2 mM DTT, 10 mM MgCl2, 50 mM NH4Cl, 1 mM spermidine, 2 mM ATP, 0.5 mM GTP, 8 mM phosphoenolpyruvate and 2 µM pyruvate kinase). Mix II (50 µg of bulk tRNA, 100 µg S100 extact and 200 pmoles of C14-Phe) was incubated at 37°C for 15 min in polyU-mix. Mix I and II were mixed and incubated at 37°C for 15 min. 14C-Phe incorporation into protein was determined by TCA precipitation and scintillation counting. All reactions were performed in triplicate.
For LacZ assays, strains carrying wild-type or mutant lacZ alleles on a plasmid were harvested in exponential phase. The assay for LacZ activity was then performed as described (Miller, 1972). The per cent LacZ activity (in Miller units) of each mutant lacZ allele relative to the wild-type lacZ allele for MC4100 or the ΔybeY mutant is reported.
We thank Dr Harry Noller for helpful discussion, Dr Murray Deutscher for his generous gifts of rnb, rnd, rph, rnt, rne-1 and rnr E. coli mutant strains and Dr Albert Dahlberg for insightful discussion and for his gift of plasmids pSG25, pSG163, pSG853, pSG3/4, plac7 and plac10. This work was supported by National Institutes of Health Grant GM31030 to G.C.W. and Grant GM17151 to U.L.R., MIT Center for Environmental Health Sciences Grant P30ES002109, a National Sciences and Engineering Research Council of Canada graduate scholarship to B.W.D. and an NCI postdoctoral fellowship to L.A.S. G.C.W. is an American Cancer Society Research Professor.