Báo cáo khoa học: Ribosome assembly in Escherichia coli strains lacking the RNA helicase DeaD⁄CsdA or DbpA

Ribosome assembly in Escherichia coli strains lacking the RNA helicase DeaD⁄CsdA or DbpA Lauri Peil, Kai Viruma¨ e and Jaanus Remme

Institute of Molecular and Cell Biology, University of Tartu, Estonia

Keywords 23S rRNA; DbpA; DeaD ⁄ CsdA; ribosome assembly; rRNA processing

Correspondence J. Remme, Riia 23, 51010 Tartu, Estonia Fax: +372 742 0286 Tel: +372 737 5031 E-mail: jremme@ebc.ee

(Received 26 February 2008, revised 22 May 2008, accepted 27 May 2008)

doi:10.1111/j.1742-4658.2008.06523.x

Ribosome subunit assembly in bacteria is a fast and efficient process. Among the nonribosomal proteins involved in ribosome biogenesis are RNA helicases. We describe ribosome biogenesis in Escherichia coli strains lacking RNA helicase DeaD (CsdA) or DbpA. Ribosome large subunit assembly intermediate particles (40S) accumulate at 25 (cid:2)C and at 37 (cid:2)C in the absence of DeaD but not without DbpA. 23S rRNA is incompletely processed in the 40S and 50S particles of the DeaD) strain. Pulse labeling showed that the 40S particles are converted nearly completely into func- tional ribosomes. The rate of large ribosomal subunit assembly was reduced about four times in DeaD-deficient cells. Functional activity tests of the ribosomal particles demonstrated that the final step of 50S assembly, the activation step, was affected when DeaD was not present. The results are compatible with the model that predicts multiple DeaD-catalyzed struc- tural transitions of the ribosome large subunit assembly.

and RsgA [16])

large

the

or

these precursor particles, suggesting involvement of proteins in ribosome assembly. A group of proteins known as ‘small GTPases’ have also been shown to participate in the assembly of the small subunit (Era subunit [10,11] (CgtAE ⁄ Obg [17,18] and EngA ⁄ Der [19]).

The E. coli genome contains five genes for DEAD- box RNA helicases: deaD, dbpA, rhlB, rhlE and srmB [20]. RNA helicase DeaD ⁄ CsdA is involved in trans- lation [21], RNA degradation [22] and ribosome assem- bly [8]. It has two names [23], but because CsdA (cold shock DEAD-box protein; named by Brandi et al. 1999 [23]) also designates the E. coli gene encoding cysteine sulfinate desulfinase (previously ygdJ-ygdK) [24], we prefer to use the original name DeaD (DEAD-box protein) [26]. Disruption of the deaD gene leads to no growth defect at 37 (cid:2)C, whereas prominent growth defects, with long adaptation periods, are observed at temperatures below 30 (cid:2)C [27]. Over- expression of RNA helicase RhlE suppresses the cold- sensitive growth defect of a strain lacking DeaD

The ribosome is a large, complex and dynamic ribonu- cleoprotein particle consisting of a large and a small subunit. In Escherichia coli, the large (50S) subunit contains two rRNA molecules (23S and 5S rRNA) and 33 ribosomal proteins (r-proteins), whereas the small (30S) subunit contains one rRNA molecule (16S rRNA) and 21 r-proteins [1]. Assembly of ribosomes is a complex and highly coordinated process, which is initiated during rRNA transcription [2] and involves processing, modification and folding of rRNA and r-proteins, as well as their association to form func- tional ribosomal subunits. Many extraribosomal fac- tors are involved in the ribosome assembly process, especially in eukaryotes [3]. In bacteria, the number of extraribosomal components so far identified as being involved in ribosome assembly is smaller [4,5]. The absence of the RNA modification enzymes RluD [6] and RlmJ [7], the RNA helicases DeaD ⁄ CsdA [8] and SrmB [9], the cold-shock protein RbfA [10,11] and the heat-shock proteins DnaK [12–14] and GroEL [15] ribosome assembly leads

to the accumulation of

Abbreviation r-protein, ribosomal protein.

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Results

Ribosomal particle content in strains deficient in DeaD and DbpA

the assembly-defective particles,

[28]. Normally, DeaD is present in small quantities in cells grown at 37 (cid:2)C, but it is induced significantly when cells are shifted to a lower temperature [27]. DeaD was shown to participate in the assembly of both ribosomal subunits. First, overexpression of DeaD is a suppressor of mutation in the small ribo- somal subunit protein S2, a mutation that blocks bind- ing of r-protein S1 [21,26]. Later, it was demonstrated that in the absence of DeaD assembly, defective ribo- somal large subunits accumulate at temperatures below 30 (cid:2)C, showing that DeaD has a function in large sub- unit biogenesis [8]. On the basis of the r-protein com- it was position of concluded that DeaD has a role during late assembly [7]. It has also been shown that DeaD is needed for the translation of highly structured mRNAs at low functional temperatures [29] and can act as a full replacement for the resident RNA helicase RhlB within the RNA degradosomes formed at low temperatures [22]. DeaD binds to numerous proteins in E. coli, including a set of r-proteins [30], and it has ATP- dependent RNA helicase activity in vitro [31].

Ribosomal particle content was analyzed by sucrose gradient centrifugation (Fig. 1). Bacteria were grown in rich medium at 25 (cid:2)C or 37 (cid:2)C, and harvested in early exponential phase (D600 nm (cid:2) 0.2), when ribo- some synthesis is most active. In the wild-type cells, ribosomes were found mostly in the 70S fraction, and, as expected, free 50S and 30S subunits were present at low levels (Fig. 1A). In contrast, DeaD- deficient (deaD414) cells had altered ribosomal parti- cle composition: the 30S fraction was increased and the 50S fraction was reduced with respect to the 30S fraction, and a new peak that sediments at around 40S appeared. A similar distribution of ribosomal particles was repeatedly observed at both tempera- tures (Fig. 1B), but the 40S peak was more pro- nounced at 25 (cid:2)C (Fig. 1B). It must be noted that at 37 (cid:2)C the 40S particles are specific to the early log growth phase and disappear at higher cell densities during mid-log growth phase (D600 nm > 0.4) (data not shown).

To control

Although several RNA helicases have been shown to participate in ribosome assembly, their exact role is not clear. It is not known whether the subribosomal particles accumulating in the DeaD deletion strain at temperatures below 30 (cid:2)C are 50S subunit assembly dead-ends or assembly precursor particles.

for whether the ribosome biogenesis defects were caused by the absence of functional DeaD protein, the deaD414 strain was transformed by the rescue plasmid pBAD–DeaD encoding wild-type DeaD protein under the control of an arabinose pro- moter (PBAD). DeaD-defective cells containing pBAD– DeaD, grown without the arabinose, had the same ribosome particle phenotype as cells without the rescue plasmid, whereas the addition of arabinose to the culture medium resulted in sufficient DeaD expres- sion to overcome the defects caused by deaD gene disruption (data not shown). Thus we conclude that the large ribosomal subunit assembly defect observed in the deaD414 strain was due to the absence of DeaD RNA helicase.

The gene

strain MG1655. The growth rate of

DbpA is unique among the E. coli RNA helicases because its ATPase activity is specifically activated by 23S rRNA [32,33]. DbpA binds to the fragment of 23S rRNA containing helix 92 [34,35]. DbpA was reported to be a weakly processive 3¢ fi 5¢ RNA helicase, which needs ATP for its activity [36]. Despite its high speci- ficity, deletion of the dbpA gene does not confer a growth defect [37]. Elles and Uhlenbeck showed recently that a mutation of the arginine finger in the active site of DbpA abolishes its ATPase and helicase activity, and gives rise to a dominant slow growth phe- notype [38]. It was suggested that DbpA is involved in ribosome assembly [23,33,39]; however, there is no experimental evidence for participation of DbpA in ribosome assembly (to our knowledge).

(Fig. 1C),

encoding RNA helicase DbpA was replaced by a chloramphenicol resistance gene in E. coli this DbpA-deficient (dbpA204) strain grown at 25 (cid:2)C and at 37 (cid:2)C was the same as for the wild-type strain MG1655 (data not shown), indicating that bacteria can grow normally in rich medium without functional DbpA. The ribosome particle composition of DbpA- deficient cells was identical to that of the wild-type bacteria at both temperatures showing ribosome assembly can proceed normally in that the absence of DbpA protein under fast growth conditions.

We have analyzed ribosome biogenesis in the E. coli strains lacking RNA helicases DbpA or DeaD. Although we cannot provide direct evidence for the involvement of DbpA in ribosome biogenesis, we have further confirmed the involvement of DeaD in ribo- some assembly. Deletion of deaD leads to the appear- ance of 40S precursor particles that are functionally inactive but capable of maturing into 50S subunits that can form 70S ribosomes, although at much lower rate than in wild-type cells.

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A

B

C

Fig. 1. Analysis of ribosome profiles of E. coli strains MG1655, deaD414 and dbpA204 strains reveals the appearance of a new 40S particle and a disturbed ratio between free 50S and 30S subunits. Cells were grown at 25 (cid:2)C or 37 (cid:2)C, and the ribo- some profile was obtained as described in Experimental procedures. MG1655 cells (A) and dbpA204 cells (C) exhibit a normal ribo- somal particle profile, where the most prom- inent peak corresponds to free 70S ribosomes and only relatively small amounts of free 50S and 30S subunits are present. deaD414 cells (B) show an altered ribosome profile, where a new particle with a sedi- mentation coefficient of approximately 40S appears.

40S and 50S particles from the DeaD-deficient strain contain 23S rRNA precursors

expected (Fig. 2A, middle panel).

The rRNA composition of the ribosomal particles was determined by RNA dot-blot analysis. Ribosomal par- ticles from both MG1655 (wild-type) and deaD414 cells grown at 25 (cid:2)C were separated in a sucrose gradi- ent (Fig. 2, upper panel) and the rRNA was purified.

70S ribosomes from wild-type cells contained both 16S and 23S rRNA, whereas free 50S subunits contained only 23S rRNA, and 30S subunits contained only 16S rRNA, as In deaD414 cells, 70S ribosomes and free 30S subunits had a RNA composition identical to that of their wild- type counterparts, whereas the 23S rRNA was found in both the 40S and 50S subunits (Fig. 2B, middle

A

B

Fig. 2. RNA analysis of sucrose gradient fractions indicates a processing defect of 23S rRNA. MG1655 (A) and deaD414 (B) cells were grown at 25 (cid:2)C, ribosome pro- files were analyzed and fractionated (upper panel), and RNA was purified as described in Experimental procedures. Purified RNA was blotted and probed with either 16S rRNA- or 23S rRNA-specific labeled oligonu- cleotide (middle panel). RNA from 23S rRNA-containing fractions was subjected to a primer extension reaction to determine the position of 23S rRNA 5¢-termini. Posi- tions corresponding to mature (+1) and pre- 23S ()3 and )7) rRNA are indicated (lower panel), together with the corresponding rrnB operon DNA sequence.

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it appears that 40S panel). According to this result, particles are derivatives of the ribosome large subunit, which is in agreement with previous reports [8].

Table 1). 40S particles from the same cells contained predominantly )7 pre-23S rRNA; )3 pre-23S rRNA and mature 23S rRNA were present as minor species (Fig. 2B, lower panel). Traces of mature 23S rRNA in the 40S particles could, in part, result from contamina- tion from the 50S fraction due to poor separation in the sucrose gradient (Fig. 2B, lower panel). In the 30S fraction, only )7 23S rRNA was found, probably due to the contamination from 40S particles. Quantitation of the 5¢-ends of 23S rRNA species was performed using densitometry of the primer extension bands cor- responding to the central fractions of the peaks in sucrose gradients, as shown in Fig. 2 (see Table 1). The processing status of 23S rRNA suggests that at least 70% of the 40S particles consist of incompletely assembled precursors of the 50S subunit, and this per- centage could even be higher if contamination with 50S particles is considered. Nevertheless, a minor frac- tion of the 40S particles contain fully processed 23S rRNA, which may represent degradation products of unstable 50S ribosomes. Similar results for the 23S rRNA processing were obtained in several independent ribosomal particle preparations, and are in qualitative agreement with the results of Charollais et al. [8].

Time course of ribosome assembly in the DeaD-deficient strain

The 40S particles from the DeaD-deficient cells could be either precursors of the 50S subunits or result from degradation of the mature 50S subunits. One way to discriminate between these two possibilities is based on the processing pathway of the 23S rRNA 5¢-end. 30S rRNA primary transcripts are first cut by RNase III, resulting in 23S rRNA species that are either three or seven nucleotides longer at their 5¢-end than the mature 23S rRNA (designated ‘)3’ and ‘)7’, respectively) [40,41], whereas the final maturation of ribosomal RNA takes place in functional 70S ribo- somes [42]. Therefore, assembly intermediate particles should contain incompletely processed 23S rRNA, whereas the degradation products derived from func- tional ribosomes will have mature 23S rRNA. Charol- lais et al. have shown that the 40S particles contain incompletely processed 23S rRNA [8]. In order to dis- criminate between assembly intermediates and degra- dation products of mature 50S subunits, quantitative analysis of 23S rRNA processing was necessary. The position of the 23S rRNA 5¢-end was analyzed with a primer extension reaction: the majority of 23S rRNA from 70S ribosomes of both strains contained mature 5¢-termini (marked as +1 in Fig. 2, lower panel) and traces of pre-23S rRNA species (marked as )3 or )7 in Fig. 2, lower panel). Free 50S subunits of both strains contained nearly equal amounts of )7, )3 and mature 23S rRNA, although the percentage of pre-23S rRNA species was increased in fractions closer to the 30S peak (Fig. 2A, lower panel; Table 1). In the 50S particles from deaD414 cells, only about one-third of the 23S rRNA was mature 23S rRNA (Fig. 2B,

We used an RNA pulse-labeling approach to measure the time course of ribosome assembly. Cells were grown at 25 (cid:2)C, and RNA was labeled for 5 min with [3H]uridine, after which the transcription initiation was blocked with rifampicin. Cells collected at different time points were lysed, ribosomes were fractionated by centrifugation in sucrose gradients, and the fractions were counted for radioactivity.

free

Table 1. Quantitative distribution of precursor and mature 23S rRNA in ribosomal particles of wild-type (MG1655) and DeaD-defi- cient strains. Ribosomal particles from cells grown at 25 (cid:2)C were fractionated by sucrose gradient centrifugation (see Fig. 2), and the 5¢-end of 23S rRNA from the top fraction of the corresponding peak was analyzed by primer extension. The relative abundance of each 23S rRNA species was determined by using scanned autoradio- graphs and IMAGEQUANT TL software (GE Healthcare Life Sciences, Little Chalfont, UK). The error was < 2%.

)7 pre-23S rRNA (%)

)3 pre-23S rRNA (%)

Mature 23S rRNA (%)

Ribosomal particles

The results of rRNA labeling in the wild-type bac- teria are shown in Fig. 3A. At the 0 min time point (when rifampicin was added to the medium), the majority of radioactively labeled RNA was found in the 30S and 50S fractions. Five minutes after the addi- tion of rifampicin, the radioactive RNA was distrib- subunit and 70S uted equally between the fractions. After 10 min, most of the radioactive signal had migrated to the 70S fraction. Further incubation did not change the distribution of radioactive RNA in sucrose gradients, from which we conclude that in the wild-type bacteria, half of the ribosomes had been assembled in 5 min and the process had been com- pleted after 10 min at 25 (cid:2)C.

MG1655 70S MG1655 50S deaD414 70S deaD414 50S deaD414 40S

9 35 11 36 48

26 33 26 29 22

65 32 63 35 30

A time course of ribosome assembly in the deaD414 cells is shown in Fig. 3B. At the 0 min time point, the majority of labeled rRNA was located in the 30S

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A

B

70S ribosomes after 20 min, and ribosome assembly was not fully completed even after 40 min (Fig. 3B). The shift of the radioactive signal from ribosomal pre- cursor particles towards mature 70S ribosomes shows that 40S particles can in fact be matured to form func- tional ribosomes, although the rate of ribosome large subunit assembly is approximately four-fold slower than in the wild-type strain. In contrast, formation of ribosomal 30S subunits is fast and proceeds without accumulation of apparent precursor particles (Fig. 3B), suggesting that small subunit assembly is not affected by the absence of DeaD. The fact that, altogether, (cid:2) 70% of the radioactive rRNA is incorporated into 70S ribosomes indicates that the degradation of assem- bly precursor particles plays a minor role, and is in agreement with the 23S rRNA processing results.

Functional activity of ribosomal particles from DeaD-deficient cells

In spite of the excess of 30S subunits in the deaD414 strain, a free 50S fraction was also clearly present in the exponentially growing cells (Figs 1 and 2). Because of poor separation of the 40S and 50S particles in the sucrose gradient, the particles were further purified by a second sucrose gradient centrifugation. Functional activity of the ribosomal particles was tested according to their ability to catalyze peptide bond formation. As peptidyl transferase activity appears during the final step of ribosomal 50S subunit assembly [43], this assay indicates whether the large ribosomal subunit assembly has gone to completion or not. In the peptidyltrans- ferase assay, [35S]fMet-tRNA was used as the donor substrate and puromycin as the acceptor. The activity of the ribosomal particles was determined at different concentrations to ensure linear concentration depen- dence. A relative activity was also calculated with respect to wild-type 50S subunits obtained by disso- ciation of mature 70S ribosomes (designated as 50S* in Table 2).

Fig. 3. Time course of ribosome assembly shows that the 40S subunits from deaD414 cells are capable of maturation into 50S subunits and 70S ribosomes. Cells were grown at 25 (cid:2)C until early exponential phase, and RNA was labeled with [3H]uridine for 5 min; after this, transcription was stopped by the addition of rifampicin. Culture aliquots were taken at different time points (0, 5, 10, 20 and 40 min; 0 min indicates the addition of rifampicin), cells were harvested, and the ribosome profile was analyzed. (A) Wild-type strain; (B) DeaD-deficient strain.

and 40S fractions. Labeled rRNA from the 40S frac- tion moved very slowly into the 70S ribosomes; only 50% of the radioactive RNA was incorporated into

50S subunits derived from 70S ribosomes from both deaD414 and wild-type cells exhibited similar levels of activity in the peptidyltransferase assay (Table 2). Free 50S subunits from the wild-type strain exhibited 2.5-fold lower activity than the mature 50S* subunits derived from 70S ribosomes (Table 2). This result suggests that (cid:2) 60% of the free 50S subunits from the wild-type strain contain newly synthesized assembly intermediate particles that sediment as 50S particles. from the deaD414 strain had Free 50S particles approximately seven-fold lower activity than the 50S* subunits and about three-fold lower activity than the free 50S particles from the wild-type strain (Table 2).

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are

functionally

50S subunits

Table 2. Functional activity of ribosomal particles according to the peptidyltransferase assay. Ribosomal particles from wild-type (MG1655) and deaD414 cells grown at 25 (cid:2)C were fractionated by sucrose gradient centrifugation (see Fig. 2). The ability of the ribo- somal particles to catalyze peptide bond formation was determined using fMet-tRNA as a donor and puromycin as an acceptor sub- strate. 50S*, particles isolated from 70S ribosomes. The error was < 6% of the activity level.

subunit

Particles

Relative activity

Strain

MG1655 MG1655 deaD414 deaD414 deaD414

50S* 50S 50S* 50S 40S

1.00 0.39 0.98 0.14 0.01

(Fig. 3). Activity tests reveal that (cid:2) 60% of the free wild-type inactive (Table 2). Taken together, these data show that the rate-limiting step of 50S biogenesis is the activation of the subunits, at both 37 (cid:2)C and 25 (cid:2)C. This fact corre- lates with the limiting heat-activation step of large reconstitution in vitro [45]. The ribosomal heat-dependent step probably reflects a conformational change of the subunit that occurs after the association of rRNA and r-proteins [43]. It is evident that rRNA undergoes post-transcriptional conformational changes [46], and it is very likely that such changes are cata- lyzed by one or more of the five RNA helicases in E. coli. Indeed, RNA helicases SrmB and DeaD have been shown to have functions in ribosome assembly [8,9,47]. Our results for ribosome assembly in DeaD- deficient cells are consistent with the findings of et al., who observed accumulation of Charollais incompletely assembled ribosomal large subunits at 25 (cid:2)C [8]. Additionally, we demonstrate that the intact DeaD protein also has a role in 50S assembly during early exponential growth phase at 37 (cid:2)C.

The time course of

These results indicate that the rate of large ribosomal strain is subunit assembly in the DeaD-deficient reduced not only in the primary assembly step, but also during the final maturation step. As expected, the 40S particles of the deaD414 strain had no activity in the peptidyltransferase assay (Table 2). Free 30S parti- cles of both the wild-type and DeaD-deficient strains exhibited equal activity during an in vitro translation assay, indicating that biogenesis of the ribosome small subunit was not affected in the absence of DeaD (data not shown).

Discussion

The E. coli genome contains five genes for DEAD-box RNA helicases. Deletion of two of them, DeaD and SrmB, leads to defects in large ribosomal subunit assembly [8,9]. It was suggested that the third RNA helicase, DbpA, may also be involved in 50S assembly [23,33]; however, to our knowledge, involvement of DbpA in ribosome assembly has not been documented. Here we demonstrate that deletion of the dbpA gene does not in fact lead to the accumulation of ribosome precursor particles under the fast growth ⁄ rich medium conditions used (Fig. 1). Although this finding shows that the RNA helicase DbpA is not essential for ribo- some assembly under these conditions, we cannot exclude the involvement of DbpA in ribosome assem- bly, as defects may appear under other growth or stress conditions.

the functional ribosomal

ribosome assembly in the deaD414 strain demonstrated that the radioactively labeled RNA eventually moves from 30S, 40S and 50S particles into 70S ribosomes, albeit at a reduced rate as compared to ribosome assembly in the wild-type strain. This result demonstrates that the 40S particles are precursors of the functional ribosomes. On the basis of the protein composition, it has been concluded in an earlier work that the 40S particles of the DeaD- deficient strain are misassembled particles rather than genuine precursors [8]. In addition, the labeling experi- ment shows that degradation of rRNA in the assembly intermediate particles is marginal. In the absence of DeaD, large ribosomal subunits require 40 min at 25 (cid:2)C to be incorporated into the 70S pool, indicating that the rate of 50S assembly is reduced four-fold as compared with the wild-type strain (Fig. 3). According to the peptidyltransferase assay, (cid:2) 90% of the free 50S subunits of the deaD414 strain were functionally inac- tive. Coupled with the fact that the level of functional 50S subunits was decreased about three-fold as com- pared to the wild-type strain (14% versus 39%; Table 1), this suggests that both the transition of 40S particles to 50S particles and the rate of the final acti- vation step of 50S subunit assembly is significantly reduced due to the absence of DeaD.

Ribosomal

Ribosome assembly in E. coli is very fast and effi- large cient. Formation of subunit occurs within 2–3 min at 37 (cid:2)C [44]. The rate-limiting step of 50S assembly is activation of the subunits, which is manifested by entering of the newly [44]. At assembled 50S subunits into the 70S pool 25 (cid:2)C, the formation of 50S subunits is fast, with func- tional 50S subunits being assembled within 5–10 min

subunit assembly defects are often accompanied by rRNA processing defects. Final matu- ration of 23S rRNA termini occurs at the level of 70S ribosomes, probably in polysomes, under protein synthesis conditions [48]. The 23S rRNA has been

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teins in the 40S particles could be due to the reduced translation level of the corresponding proteins. If this is true, the accumulation of 40S particles is caused by the lack of specific r-proteins. Therefore, if RNA heli- case DeaD functions during translation initiation of r-proteins, its absence would lead to a imbalance of r-protein production, which in turn would cause ribo- some assembly defects. However, such indirect inhibi- tion of ribosomal subunit assembly does not preclude direct participation of DeaD in the ribosome assembly process. One important observation in favor of such a direct involvement is the association of the DeaD pro- tein with assembly intermediate particles [8]. In addi- tion, the fact that the final activation step of the 50S subunit, after association of all large subunit proteins, is adversely affected in the deaD414 cells indicates that at least one assembly step depends on DeaD, a step that is clearly distinct from r-protein synthesis.

that 40S particles

In conclusion, RNA helicase DeaD participates in multiple steps of ribosome assembly, most likely by facilitating different RNA conformational transitions that are necessary during this complex process.

Experimental procedures

found to be incompletely processed in large ribosomal subunit precursor particles from a variety of E. coli deletion strains, i.e. those lacking SrmB [9], RrmJ ⁄ FtsJ [7], CgtAE ⁄ ObgE [17], EngA ⁄ Der [19], and RluD [6]. 23S rRNA precursors have been found in 40S particles of DeaD-deficient strain [8], which is in good agree- ment with our work. Interestingly, the ratio of pre-23S rRNA molecules carrying three or seven extra nucleo- tides at the 5¢-end is different in the 40S assembly intermediate particles and large subunits from the deaD414 strain (Table 1), indicating the different origin of precursor particles and suggesting that there are multiple possible assembly check-points. Multiple assembly pathways found during in vitro reconstitution of 30S subunits [49] support this idea. Consistently, RNase III has been shown to act on the naked pre- 23S rRNA to produce a )7 species of 23S rRNA, as well as on the pre-23S rRNA in the 50S subunit to produce a )3 species [41]. Therefore, we conclude that )3 and )7 pre-23S rRNA represent RNase III process- ing products formed during different stages of ribo- from some assembly. The fact DeaD-deficient cells contain mostly )7 pre-23S rRNA suggests that the processing event occurs during early assembly, when only few proteins are associated with the rRNA.

Bacterial strains and plasmids

It was recently found that

the 23S rRNA from deaD414 40S and 50S particles is incompletely modi- fied, lacking three pseudouridines at positions 1911, 1915, and 1917, made by pseudouridine synthase RluD [50]. This shows that RluD is a late assembly-specific enzyme. It is not likely that DeaD helicase is directly involved in rRNA modification, as the 23S rRNA in the 70S ribosomes has RluD-specific modifications independently of the presence of DeaD protein. Instead, we think that the DeaD helicase helps to fold 23S rRNA into a conformation that is a substrate for RluD. The absence of specific modifications in 23S rRNA from 50S particles further supports the conclu- sion that RNA helicase DeaD participates in the final steps of large ribosomal subunit assembly.

into MG1655 ⁄ pKD46-competent

translation of

structured mRNAs

The E. coli strain MG1655 [53] was used as parental strain. The in-frame deletion of the DbpA gene, giving the dbpA::- cat strain (dbpA204), was performed according to the method of Datsenko & Wanner [54]. The cat cassette from the pKD3 plasmid was amplified by PCR, using Pwo poly- merase (Boehringer Mannheim) and primers dbpA204::- cat(pKD3) ⁄ 5¢ (5¢-CCA TGA CGC CGG TGC AGG CCG CCG CGC TTC CGG CGA TCC GTG TAG GCT GGA GCT GCT TC-3¢) and dbpA204::cat(pKD3) ⁄ 3¢ (5¢-GAA TAA GAT GTT TAC TCT TGC ACC CGG CAA TTC AAC ATT TCA ATG GGA ATT AGC CAT GGT CC-3¢) (complementary regions to the dbpA gene or its flanking regions are underlined). The resulting 1114 bp PCR prod- uct was gel-purified using an UltraClean 15 DNA Purifica- tion kit (MO BIO Laboratories, Carlsbad, CA, USA), and cells, electroporated which were previously grown in the presence of 10 mm arabinose and made competent by concentrating 10-fold and washing five times with ice-cold 10% glycerol. Selection for the recombination event and elimination of the pKD46 plasmid were performed as described previously [54]. Colo- nies were tested for the dbpA deletion by PCR, using prim- ers flanking the gene, and by Southern blot analysis. The deaD414 strain,

in which the deaD gene was dis- rupted by a kanamycin resistance cassette, was a generous gift from K. E. Rudd (University of Miami School of Medi- cine, FL, USA). For our experiments, bacteriophage P1

The absence of the deaD gene product has been shown to alter translation of specific mRNAs in vivo [27], and in a cell-free system DeaD has been shown to stimulate [29]. mRNAs encoding r-proteins often have complex sec- ondary structure around the initiation region, which is used to regulate translation by a competition mecha- nism [51,52]. A specific set of large ribosome subunit proteins has been found in reduced amounts in 40S particles [8]. Strong reduction of polysomes has been observed in the absence of DeaD [8], suggesting a reduced translation level. Thus, the absence of r-pro-

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transduction was used to transfer the deaD::kan gene into E. coli strain MG1655 [50].

For the rescue plasmid, the deaD gene was amplified by PCR using primers 5¢-GCC CAT GGC TGA ATT CGA AAC CAC TTT TG-3¢ and 5¢-CGG GAT CCT TAC GCA TCA CCA CCG AAA CG-3¢ (the NcoI and BamHI sites are underlined). Gel-purified PCR products were cut with NcoI and BamHI, gel-purified again, and ligated with pBADMy- cHisC plasmid (Novagen, Madison, WI, USA) cut with NcoI and BglII, resulting in the plasmid pBAD–DeaD.

Ribosome profile analysis

fractions were dot-blotted onto a Hybond N+ membrane (Amersham Biosciences, Little Chalfont, UK), using vacuum manifold and according to the manufacturer’s protocol. Two parallel membranes were prepared, one to be probed with a 5¢-labeled 16S RNA-specific probe (5¢-GCCAGCGTTCAATCTGAG-3¢) and another with a 23S RNA-specific probe (5¢-GCTTTCTTTAAATGATGG CTGCTT-3¢). RNA was UV-crosslinked, and hybridization was performed as follows. Prehybridization was performed at 50 (cid:2)C for 6 h in 25 mL of hybridization solution (5· SSC, 5· Dennhardt’s solution [55], 0.5% SDS, carrier DNA), after which radioactively labeled oligonucleotides were added to the hybridization solution. Hybridization was performed at 50 (cid:2)C for 12 h, after which membranes were washed twice for 5 min with 2· SSC and 0.1% SDS at 50 (cid:2)C, and twice for 10 min with 1· SCS and 0.1% SDS at 50 (cid:2)C. Washed membranes were visualized by auto- radiography.

The 5¢-end of 23S rRNA was analyzed on two to four different rRNA preparations of each strain using a primer extension reaction as described previously [56].

rRNA pulse labeling

Cells were grown in 200 mL of 2 · YT medium to a D600 nm of 0.2–0.3. Expression from pBAD plasmids was induced with 1 mm arabinose. Cells were rapidly cooled, collected by centrifugation at 2800 g for 8 min, and resus- pended in 1 mL of ice-cold buffer LP [16% (w ⁄ w) sucrose, 50 mm Tris ⁄ HCl (pH 8.0), 60 mm KCl, 60 mm NH4Cl, 6 mm MgOAc, 6 mm b-mercaptoethanol]. After addition of freshly prepared lysozyme (final concentration 1 mgÆmL)1 in water; Sigma, St Louis, MO, USA) and RNase-free DNase I (final concentration 20 UÆmL)1; Amresco, Solon, OH, USA), cells were lysed by three freeze–thaw cycles. After the third freeze–thaw cycle, the lysate was clarified by centrifugation in a microfuge at 18 000 g for 20 min at 4 (cid:2)C. Clarified lysate was diluted with an equal volume of ice-cold buffer LLP [10 mm Tris ⁄ HCl (pH 8.0), 60 mm KCl, 60 mm NH4Cl, 12 mm MgOAc, 6 mm b-mercaptoeth- anol], and 2 mL of diluted lysate (80–130 A260 nm units) was layered onto a 10–25% (w ⁄ w) sucrose gradient in buf- fer LLP and centrifuged for x2t = 2.7 · 1011 at 4 (cid:2)C in a Beckman SW28 rotor. Gradients were analyzed with an LKB Uvicord detector with continuous monitoring at 254 nm, and fractions were collected if needed. The experi- ment was repeated for each strain 6–12 times.

For RNA pulse-chase experiments, cells were grown in 400 mL of modified LB medium (10 gÆL)1 tryptone, 1 gÆL)1 yeast extract, 10 gÆL)1 NaCl) at 25 (cid:2)C to an D600 nm of 0.2. RNA was labeled with 20–30 lCi (wild-type strain) or 20–60 lCi (deaD414 strain) of [5,6-3H]uridine (Amersham Biosciences) for 5 min, after which the initiation of RNA transcription was stopped by the addition of rifampicin (final concentration 500 lgÆmL)1). Eighty-milliliter aliquots of culture were taken at different time points (0, 5, 10, 20 and 40 min; 0 min indicates the addition of rifampicin), and cells were cooled rapidly on ice, collected by centri- fugation at 2800 g for 8 min, and suspended in 1 mL of ice-cold LP buffer, containing 1 mgÆmL)1 lysozyme and 20 UÆmL)1 DNase I.

RNA analysis

Ribosome profile analysis was performed as described above, except that the area covering 70S ribosomes and free ribosome subunits was fractionated into 40 fractions. RNA was precipitated with an equal volume of ice-cold 20% tri- chloroacetic acid, radioactive RNA was collected into 25 mm diameter glass-fiber membranes (Millipore, Billerica, MA, USA), and radioactivity was counted after the addi- tion of OptiPhase HiSafe liquid scintillator (PerkinElmer, Waltham, MA, USA), using a WinSpectral 1440 (Wallac, Turku, Finland) liquid scintillation counter. rRNA pulse labeling experiments were repeated three times with the deaD414 strain and six times with MG1655.

For RNA analysis, ribosomal particles were separated by sucrose gradient centrifugation, and 1 mL fractions were collected, precipitated with ethanol, and dissolved in 200 lL of LLP buffer. Then, 1 mL of 5 m GuSCN ⁄ 4% Triton X-100 was added, and the tubes were shaken for 20 min at room temperature. RNA was bound by adding 20 lL of silica (50% suspension in water), and the tubes were shaken for another 10 min. After brief centrifugation at 10 000 g for 30 s, the supernatant was discarded and the pellet was resuspended in 1 mL of 5 m GuSCN and shaken for 10 min, after which two successive rounds of washing with 50% ethanol followed. Finally, RNA was eluted with 50 lL of water, and A260 nm was measured.

Peptidyltransferase assay

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Functional activity of the 50S subunits was assayed acc- ording to their ability to catalyze peptide bond formation. For RNA dot-blot analysis, 0.1 pmol of RNA from the topmost gradient fraction (containing 50S ribosome sub- units) and equal volumes of RNA from other gradient

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DbpA and DeaD ⁄ CsdA in ribosome assembly

substrate was

6 Gutgsell NS, Deutscher MP & Ofengand J (2005) The pseudouridine synthase RluD is required for normal ribosome assembly and function in Escherichia coli. RNA 11, 1141–1152. 7 Bugl H, Fauman EB, Staker BL, Zheng F, Kushner

SR, Saper MA, Bardwell JC & Jakob U (2000) RNA methylation under heat shock control. Mol Cell 6, 349– 360.

8 Charollais J, Dreyfus M & Iost I (2004) CsdA, a cold- shock RNA helicase from Escherichia coli, is involved in the biogenesis of 50S ribosomal subunit. Nucleic Acids Res 32, 2751–2759. 9 Charollais J, Pflieger D, Vinh J, Dreyfus M & Iost I liquid scintillator

(2003) The DEAD-box RNA helicase SrmB is involved in the assembly of 50S ribosomal subunits in Escherichi- a coli. Mol Microbiol 48, 1253–1265. conditions described same the as

10 Inoue K, Alsina J, Chen J & Inouye M (2003) Suppres- sion of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era, an essential GTPase in Escherichia coli. Mol Microbiol 48, 1005–1016. 11 Inoue K, Chen J, Tan Q & Inouye M (2006) Era and

[35S]fMet-tRNA (3.2 pmol, The donor 22 000 d.p.m.) and the acceptor was puromycin (0.4 mm final concentration). The reaction was started by adding 40% methanol to the large ribosomal subunits (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2 or 4 pmol) in buffer LLP (10 mm Tris ⁄ HCl, pH 8.0, 60 mm KCl, 60 mm NH4Cl, 12 mm MgOAc, 6 mm b-mer- captoethanol), and samples were incubated for 30 min at 30 (cid:2)C. The reaction was stopped by the addition of 10 lL of 10 m KOH and incubation for 15 min at 42 (cid:2)C. [35S]fMet- puromycin was extracted to the organic phase by addition of 1 mL of ethyl acetate and vigorous shaking. Radioactivity was counted in 0.5 mL of the organic phase with a WinSpec- tral 1440 (Wallac) liquid scintillation counter, using Opti- (PerkinElmer). Three Phase HiSafe independent preparations of 40S and 50S particles were puri- fied by two consecutive sucrose gradient centrifugations under above (x2t = 2.7 · 1011 at 4 (cid:2)C). 50S* particles were obtained by dissociating 70S ribosomes in 1 mm MgCl2 buffer and then using sucrose gradient centrifugation under the same condi- tions as described above (x2t = 2.7 · 1011 at 4 (cid:2)C). Relative peptidyltransferase activity was calculated from the linear part of the ribosome concentration blot. RbfA have overlapping function in ribosome biogenesis in Escherichia coli. J Mol Microbiol Biotechnol 11, 41–52.

Acknowledgements

12 Alix JH & Guerin MF (1993) Mutant DnaK chaper- ones cause ribosome assembly defects in Escherichia coli. Proc Natl Acad Sci USA 90, 9725–9729. 13 El Hage A & Alix JH (2004) Authentic precursors to

ribosomal subunits accumulate in Escherichia coli in the absence of functional DnaK chaperone. Mol Microbiol 51, 189–201.

We thank Dr Daniel Wilson (University of Munich) for suggestions and for correcting the English, Dr Tanel Tenson and Dr U¨ lo Maiva¨ li for critically read- ing the manuscript, and Dr Aivar Liiv (all from University of Tartu) for help and discussions. The research was supported by Estonian Science Founda- tion Grants Nos 5822 and 7509.

14 Alix JH & Nierhaus KH (2003) DnaK-facilitated ribo- some assembly in Escherichia coli revisited. RNA 9, 787–793.

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