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Journal of Bacteriology, February 2003, p. 983-990, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.983-990.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Escherichia coli Endoribonucleases Involved in Cleavage of Bacteriophage T4 mRNAs

Yuichi Otsuka, Hiroyuki Ueno, and Tetsuro Yonesaki^*

Department of Biology, Graduate School of Science, Osaka University, Osaka 560-0043, Japan

Received 8 August 2002/ Accepted 11 November 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The dmd mutant of bacteriophage T4 has a defect in growth because of rapid degradation of late-gene mRNAs, presumably caused by mutant-specific cleavages of RNA. Some such cleavages can occur in an allele-specific manner, depending on the translatability of RNA or the presence of a termination codon. Other cleavages are independent of translation. In the present study, by introducing plasmids carrying various soc alleles, we could detect cleavages of soc RNA in uninfected cells identical to those found in dmd mutant-infected cells. We isolated five Escherichia coli mutant strains in which the dmd mutant was able to grow. One of these strains completely suppressed the dmd mutant-specific cleavages of soc RNA. The loci of the E. coli mutations and the effects of mutations in known RNase-encoding genes suggested that an RNA cleavage activity causing the dmd mutant-specific mRNA degradation is attributable to a novel RNase. In addition, we present evidence that 5'-truncated soc RNA, a stable form in T4-infected cells regardless of the presence of a dmd mutation, is generated by RNase E.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The dmd gene of bacteriophage T4 plays a role in the regulation of mRNA stability in a stage-dependent manner, discriminating between mRNA species (22). When a mutant dmd gene infects Escherichia coli cells at low temperatures, late genes are globally silenced because of the rapid degradation of their mRNAs (6). Because the dmd mutant-specific degradation of late-gene mRNA (dmdDL) is activated during T4 infection (7, 22), this activity seemed to be encoded by T4. Previously, we isolated seven pseudorevertants of a dmd mutant, which contained extragenic suppressors ssf1 through ssf7, derived from five different T4 genes (7; unpublished data). Except for ssf4, these suppressors have only weak effects on the stability of late-gene mRNA and the growth of dmd mutants. ssf4 is a strong suppressor but is unstable upon genetic manipulation (7). Recent analysis revealed that ssf4 segregated a weak suppressor in a cross with a dmd mutant, suggesting that the ssf4 strain contains multiple mutations. Thus, we were unable to isolate any single suppressor mutations strong enough to account for dmdDL. Thus, we began to doubt that T4 per se encodes an activity responsible for dmdDL. An alternative possibility is that host cells encode such an activity.

Previously, we identified an activity introducing dmd mutant-specific cleavages into RNA, presumably causing dmdDL. This activity exhibited a significant feature. Some of the dmd mutant-specific cleavages occurred only when the target region was translatable or downstream of a termination codon (8), suggesting that these cleavages were translation dependent. The other cleavages were apparently independent of translation. If host cells encode such an activity, then we might be able to detect dmd mutant-specific cleavages of RNA even in uninfected cells. In the present study, we detected dmd mutant-specific cleavages of T4 late-gene soc RNA in uninfected cells. In addition, we isolated E. coli mutants in which a dmd mutant was able to grow. One of these completely suppressed the dmd mutant-specific cleavages of soc RNA and also fully supported the growth of a dmd mutant. These results strongly suggest that the mRNA-degrading activity causing dmdDL is encoded by the host.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phages and bacterial strains. Wild-type bacteriophage T4 is T4D. The amSF16 phage contains an amber mutation in the dmd gene (6, 22). The amSF16 soc-nel phage has been described previously (8). Phage GT7 was used for transduction (27). E. coli strains used in this study are listed in Table 1. Strain TY9114 was constructed by GT7-mediated transduction of a kanamycin resistance marker from strain DK533 (20). Strains TY0225, TY0483, TY1723, TY1798, TY2134, and TY2423 were constructed by plasmid displacement using pKmiscR (see below).

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TABLE 1. Bacterial strains used in this study

Plasmids. Plasmids pTK40, pTK50, pTK61, pTK70, and pTK80, carrying soc alleles soc⁺, soc-als, soc-sls, soc-nel, and soc-hlf, respectively, have been described previously (8). In this study, we recloned the soc alleles soc⁺, soc-als, and soc-sls to construct pTK42, pTK52, and pTK60, respectively, so that all alleles were cloned in pBluescript II (Stratagene) in the same direction as the lac promoter. Plasmid pKm7 (21) containing iscR::kan was a kind gift of Y. Takahashi, Osaka University. To clone iscR::kan without flanking sequences outside iscR, the relevant DNA segment was amplified by PCR with pKm7 as a template and primers (5'-GTTTACGGAGTATTTAGCAC and 5'-GCCTGATGCGACGCGTAATG) set within the coding region of this gene. Then the DNA segment was cloned into the SmaI site in vector pKO3 (13) to construct pKmiscR.

Construction of strains with iscR disruption. Following transformation with pKmiscR, chromosomal iscR disruptants were isolated according to the method described by Link et al. (13). Briefly, transformants were plated at 42°C, a temperature nonpermissive to the pKO3 replicon, in order to select chromosomal integrants of the plasmid. Subsequently, integrants were appropriately diluted and plated at 30°C on Luria-Bertani (LB) plates containing 5% sucrose to select cells that had lost the plasmid. Finally, sucrose-resistant, kanamycin-resistant, and chloramphenicol-sensitive colonies were screened. The candidates were examined for insertion of kan in iscR by PCR with primers set to sequences outside iscR. Isolated iscR disruptants are listed in Table 1.

Isolation of host std mutants. MH1 cells (Table 1) were grown to 5 x 10⁸ cells/ml in LB medium, harvested by centrifugation, and suspended in a buffer consisting of 0.01 M potassium phosphate (pH 7.2) and 0.14 M sodium chloride. Cells were UV irradiated for 2.5 min at 1.0 J m^-2 s^-1 to yield a survival of 0.5%. Clones raised after UV irradiation were picked with a sterile toothpick and transferred into 100 µl of LB medium. These were incubated at 30°C for 3 h, and 5 µl of each was spotted on an LB plate and dried. Then 1 µl of a solution containing dmd mutant phage at 10⁶ PFU/ml was spotted onto each bacterial spot. Plates were incubated overnight at 30°C for examination of the growth of the dmd mutant.

Bacterial conjugation. Recipient std mutants were transformed with a derivative of pBR322, the tet gene of which was disrupted by an insertion at the HindIII site. Recipient and donor Hfr cells were grown to early-log phase in LB broth and mixed at a recipient-to-donor ratio of 10. After standing at 37°C for 40 min, the cells were quickly cooled on ice, and conjugal bridges were sheared by rapidly passing the cells several times through a syringe needle. An aliquot was plated onto an LB plate supplemented with ampicillin to kill the donors and with either kanamycin or tetracycline as a selective drug. Colonies raised after overnight incubation at 37°C were tested for ability to support the growth of a dmd mutant as described above.

RNA analysis. Cells were grown to a density of 5 x 10⁸/ml at 30°C in M9 minimal medium supplemented with 0.3% Casamino Acids, 1 µg of thiamine/ml, and 20 µg of tryptophan/ml. To prepare RNA from uninfected cells, a 1.5-ml culture was quickly chilled on ice and cells were harvested by centrifugation. To prepare RNA from T4-infected cells, cells were infected at 30°C with T4 at a multiplicity of 7. At various times after infection, a 1.5-ml sample was quickly chilled on ice and the cells were harvested by centrifugation. Total RNAs were extracted according to the method of Kai et al. (6). Northern blot and primer extension analyses of soc RNA were performed as described previously (8). Primer 2, described in the previous study (8), was used for primer extension.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cleavage of soc RNA in uninfected cells. In T4-infected cells, transcription of the soc gene is initiated from its own late promoter by a modified RNA polymerase in which ⁷⁰ is replaced with a T4-specific sigma factor encoded by gene 55 (26). However, we found by computer analysis that an E. coli promoter-like sequence overlapped with the soc late promoter (Fig. 1). In fact, when the soc gene along with the promoter region was cloned into the multiple cloning site of pBluescript II, it could be transcribed in uninfected cells regardless of its orientation relative to the lac promoter. The transcription started from two different points: +1 (the same as in T4-infected cells) and -2 (data not shown). A previous study identified the dmd mutant-specific cleavages of soc RNA (8). In order to determine if an activity responsible for such cleavages is encoded by E. coli, we transformed MH1 cells with plasmid pTK42 carrying soc⁺ and analyzed the cleavage of soc RNA in uninfected cells by primer extension.

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FIG. 1. Promoter region of the soc gene. The T4 late promoter is boxed. An E. coli promoter is marked in the -10 and -35 regions. Two transcription start sites are indicated by arrows.

Total RNA extracted from MH1 cells harboring pTK42 was used for cDNA synthesis, and products were resolved by electrophoresis through a sequencing gel (Fig. 2). The band marked F corresponds to full-length soc transcripts starting at positions -2 and +1. In T4-infected cells, soc RNA was processed into a stable species that underwent a truncation of 59 nucleotides at its 5' terminus relative to full-length soc RNA (8). In the present study, we also detected a cDNA band marked T, which corresponds to the 5'-truncated soc RNA. This result suggested that the 5' truncation was attributable to a host activity (see "Attribution of the 5' truncation of soc RNA to RNase E" below). In addition, along with many others, cDNAs marked TC1, TC2, and TU were discernible (Fig. 2, lane 1). These bands were identical to those identified in cells infected with a T4 dmd mutant. Cleavages at TC1 and TC2 were suggested to be introduced during peptide chain elongation, while the cleavage at TU occurred independently of translation (8).

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FIG. 2. Primer extension analysis of soc RNA from uninfected cells. Total RNA from MH1 cells carrying a plasmid-borne soc allele was extracted and used as a template for primer extension. The soc alleles were as follows: wt, wild type; als, soc-als; sls, soc-sls; nel, soc-nel; hlf, soc-hlf. Labeled cDNAs were analyzed by electrophoresis through a 5% polyacrylamide gel containing 7 M urea. Bands F and T, which were detected with all soc alleles, correspond to full-length and 5'-truncated soc RNAs, respectively. Bands NE, HL, TC1, TC2, and TU are identical to those specific to dmd mutant-infected cells (see the text for details). A set of sequence ladders for wild-type soc obtained by the dideoxy-termination method with the same primer were run in parallel.

In order to further characterize the cleavages of soc RNA in uninfected cells, we examined various soc alleles. soc-als and soc-sls are untranslatable because the initiation codon or the Shine-Dalgarno sequence is disrupted. soc-nel and soc-hlf have a premature termination codon at codons 2 and 41, respectively, and these termination codons are located upstream of TC1 and TC2 (8). In agreement with the results obtained for dmd mutant-infected cells, cleavages at TC1 and TC2 were not detected with soc-als (Fig. 2, lane 3), soc-sls (lane 2), soc-nel (lane 4), or soc-hlf (lane 5) in uninfected cells, while a cleavage at TU was detected with all the soc alleles. Furthermore, termination codon-dependent cleavages at NE and HL were detected with soc-nel (Fig. 2, lane 4) and soc-hlf (lane 5), respectively. These results indicate that the cleavages of soc RNA in uninfected cells shared all characteristics with those in dmd mutant-infected cells, strongly suggesting that an activity responsible for the dmd mutant-specific cleavages of soc RNA is encoded by E. coli.

Effects of known host endoribonucleases on the growth of a T4 dmd mutant. Because RNA cleavages identical to the dmd mutant-specific cleavages were produced in uninfected cells, we attempted to identify the endoribonuclease(s) responsible for such cleavages. At present, five endoribonucleases are known or suggested to cleave mRNAs in vivo: RNase I* (an unprocessed form of the periplasmic enzyme RNase I), RNase III, RNase E, RNase G, and RNase P (1, 2, 4, 5, 15, 17, 18, 23). The dmd mutant has a severe defect in growth because of dmdDL, and the specific cleavages of RNA may cause dmdDL (6, 8). If one of the known endoribonucleases is responsible for the dmd mutant-specific mRNA cleavages, then dmd phage would be able to grow in its absence. Therefore, we examined burst sizes (number of progeny per infected cell) to test the effects of these RNases on the growth of a dmd mutant (Table 2).

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TABLE 2. Growth of a dmd mutant on E. coli mutants defective in known RNasesa

Among these endoribonucleases, RNase E has the most prominent effect on mRNA metabolism, and therefore it is considered the primary endoribonuclease for mRNA decay in E. coli. This enzyme is also involved in the processing of rRNA. rne encodes RNase E, and the rne-131 allele has a frameshift mutation at the 585th codon, which removes the arginine-rich RNA binding domain and the degradosome scaffold region (9). This mutation stabilizes individual mRNAs as well as bulk mRNA, although it does not affect the processing of rRNA (9, 14). When wild-type phage infected SH3208 (rne⁺) or Bz215 (rne-131) cells, there was virtually no difference in the burst sizes. The dmd mutant grew poorly on SH3208 cells and produced a burst size of only 0.1. The burst size obtained with Bz215 cells was also 0.1. Thus, the rne131 mutation affected the growth of neither the dmd mutant nor the wild-type phage. Similarly, we observed no significant effect on T4 growth of mutations in rna (RNase I), rnc (RNase III), rng (RNase G), or rnp (RNase P). From these results, it is unlikely that any of the known host RNases is responsible for the dmd mutant-specific RNA cleavages.

Host mutants suppressing the growth defect of a dmd mutant. If an unknown RNase is responsible for the dmd mutant-specific mRNA cleavages, it would be important to identify it. As an initial step, we attempted to isolate E. coli mutants that could suppress the growth defect of a dmd mutant. MH1 cells were UV irradiated, and surviving clones were examined for the ability to support the growth of a dmd mutant. Out of 2,688 clones tested, we obtained 5 such mutants and named their mutations std-1 through std-5 (suppressor of T4 dmd mutant). The dmd mutant exhibited an efficiency of plating of less than 10^-5 on parental MH1 cells. In contrast, it grew well on all five host mutants, with an efficiency of plating of nearly 1 (Fig. 3). The dmd mutant grew very poorly on MH1 cells, with a burst size only 0.5% of wild-type phage. On the other hand, it produced burst sizes the same as wild-type phage on cells carrying std-2. The burst sizes of the dmd mutant on other host mutants were also remarkably higher than on MH1 cells; the burst sizes on cells carrying either std-1, std-3, or std-4 were 1/10, and that on cells carrying std-5 was about 1/2, of the wild-type level (Table 3). Therefore, suppression of the growth defect of the dmd mutant was complete for std-2 and partial for other std mutants. None of these host mutants significantly affected the growth of wild-type phage.

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FIG. 3. Effects of host mutations on the growth of the dmd mutant. A solution containing wild-type phage or the dmd mutant was serially 10-fold diluted, and 2 µl containing the number of phages indicated on the left was spotted onto a plate seeded with MH1 (std+), TY0224 (std-1), TY0482 (std-2), TY1722 (std-3), TY1797 (std-4), or TY2133 (std-5) cells. Photographs were taken after overnight incubation at 30°C.

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TABLE 3. Effects of host mutations on the growth of the dmd mutant

Suppression of the growth defect of a dmd mutant should accompany stabilization of late-gene mRNAs. To confirm this idea, we investigated soc mRNA, because its degradation has been extensively characterized (8). Rifampin was added to T4-infected cells at 21 min after infection, and total RNAs were prepared at 26, 29, 32, 35, and 46 min to analyze the decay rate of soc mRNA by Northern blotting (Fig. 4). The half-life (t_1/2) of soc mRNA was 40 min in wild-type phage-infected MH1 cells, while it was 2.3 min in dmd mutant-infected MH1 cells. As expected, when the dmd mutant-infected cells carried std-2, the half-life of soc mRNA was 40 min, equal to that in wild-type-infected MH1 cells. On the other hand, when the dmd mutant-infected cells carried std-3, the half-life of soc mRNA was 12.3 min. These results indicate that dmd mutant-specific soc mRNA degradation is completely suppressed by std-2 and partially suppressed by std-3.

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FIG. 4. Decay of soc mRNA in T4-infected cells. MH1, TY0482, or TY1722 cells were grown and infected at time zero with wild-type phage or the dmd mutant as described in Materials and Methods. Rifampin was added to cell cultures at 21 min to a final concentration of 200 µg/ml, total RNAs were extracted at 26, 29, 32, 35, and 46 min, and 2.5 µg of each RNA was electrophoresed through a polyacrylamide gel and analyzed by Northern blotting. Bands F and T, full-length and 5'-truncated soc RNA, respectively. The t1/2 of soc mRNA, shown at the bottom, was determined by measuring the signal intensity of band F at each time point.

The 5'-truncated soc RNA, indicated by T in Fig. 4, is presumably produced by processing a transcript initiated from a middle promoter located 1.2 kb upstream of soc (8) (see "Attribution of the 5' truncation of soc RNA to RNase E" below). This species was stable regardless of dmd mutation (t_1/2 = 30 min). The amount of this species was somewhat variable from experiment to experiment, but we observed no correlation with dmd mutation (8). As seen in Fig. 4, the std mutations did not affect the stability of the 5'-truncated RNA, suggesting that the endoribonuclease causing the instability of soc mRNA in a dmd mutant is different from that involved in the 5' truncation of soc RNA.

Next, we examined the effects of host mutations on cleavages of soc RNA by primer extension analysis, using total RNAs from T4-infected cells at 21 min after infection (Fig. 5). The dmd mutant-specific cleavages of wild-type soc RNA were represented by specific cDNAs TC1, TC2, and TU (Fig. 5; compare lanes 1 and 2). The soc-nel allele-specific band, NE, was also associated with the dmd mutant (Fig. 5, lane 3). As expected, with RNA from dmd mutant-infected cells carrying std-2, the specific cleavages were not detected (Fig. 5, lanes 4 and 5). By use of RNA from dmd mutant-infected cells carrying std-3, cDNAs corresponding to the specific cleavages were detected, but their intensities appeared relatively weak in comparison to those obtained by use of RNA from dmd mutant-infected MH1 cells (data not shown).

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FIG. 5. Primer extension analysis of soc transcripts from T4-infected cells. MH1 and TY0482 cells were infected with T4 carrying soc+ (wt) or soc-nel (nel) with or without a dmd mutation. RNAs were extracted 21 min after infection and used as templates for primer extension. Bands F and T correspond to full-length and 5'-truncated soc RNAs, respectively. Bands NE, TC1, TC2, and TU are specific to the dmd mutation (see the text for details).

Mutation loci. The host mutants were conjugated with nine Hfr strains with an F factor inserted at different locations on the E. coli genome (Table 1). Because these Hfr strains had a transposon, Tn10, 20 to 30 min downstream of each F factor, recombinants were selected by using the tetracycline resistance of Tn10 (see Materials and Methods) and examined for the ability to support the growth of a dmd mutant. Conjugation of cells carrying either std-2, std-3, or std-5 with srlD::Tn10 yielded 80% of recombinants that could not support the growth of the dmd mutant. Conjugation of these std mutants with zed-977::Tn10 cells yielded 30% of recombinants that could not support the growth of the dmd mutant. On the other hand, conjugation with other Hfr strains yielded less than 5% of recombinants that were unable to support the growth of the dmd mutant. These results suggested that the mutation loci of std-2, std-3, and std-5 were located between 55 and 61 min on the E. coli genomic map. Next, we used T4 GT7 phage-mediated transduction (27). GT7 phage stocks were prepared on a strain carrying a drug resistance gene located at 57.1 (iscR::kan), 59.2 (ssrA::kan), or 60.9 (srlD::tet) min and were used to infect cells carrying an std mutation. After infected cells were plated on media supplemented with an appropriate drug, colonies were examined for the ability to support the growth of a dmd mutant as a measure of linkage of each std locus to the locus inserted by a drug resistance marker. Two std loci, std-2 and std-5, showed significant linkage (60%) with the ssrA locus, while they showed no significant linkage with the iscR and srlD loci. In contrast, std-3 showed no significant linkage with the ssrA locus. Instead, all the transductants (96 of 96 clones tested) that received a drug resistance marker from iscR::kan were unable to support the growth of the dmd mutant. This result suggested that the std-3 mutation was very close to or inside of iscR. Nevertheless, we investigated an alternative possibility, that the effect of std-3 on a dmd mutant required the iscR gene (see below).

Requirement of iscR for the effects of std-1, -3 and -4. iscR is part of the isc operon and encodes a transcriptional repressor of this operon (19). Previously, we found that a dmd mutant was able to grow normally on cells that had been transformed with a plasmid carrying iscR (unpublished data). Therefore, the strong linkage of std-3 with iscR suggested that the effects of std-3 emerged via iscR or that std-3 causes overexpression of the iscR gene (see Discussion). In order to investigate this possibility, we first sequenced the iscR gene of cells carrying std-3 and found no alterations in its coding region (data not shown). Then we displaced iscR in cells carrying std-3 with iscR::kan. For this manipulation, we took special care to preserve the sequence outside of the iscR coding region: only the sequence within the iscR coding sequence was cloned as flanking sequences in a vector, pKO3, for gene replacement (see Materials and Methods). Figure 6 shows the result with iscR disruptant cells carrying std-3. The dmd phage grew on the cells with std-3 but not when the cells carried the additional iscR::kan, indicating that the effect of std-3 on the growth defect of the dmd mutant required iscR. We also similarly examined other host mutants for such an iscR requirement. The results demonstrated that the suppressive effects of std-1 and std-4 also require iscR, while those of std-2 and std-5 cells do not.

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FIG. 6. Requirement of iscR for the suppressive effect of host mutants. A solution containing dmd mutant phages was spotted in the same manner as in the experiment for which results are shown in Fig. 3. The strains used as indicators were, from left to right, MH1, TY2423, TY0224, TY0225, TY0482, TY0483, TY1722, TY1723, TY1797, TY1798, TY2133, and TY2134, and their std and iscR alleles are shown at the top. Photographs were taken after overnight incubation at 30°C.

Attribution of the 5' truncation of soc RNA to RNase E. The 5' truncation of soc RNA originally found in T4-infected cells also occurs in uninfected cells (Fig. 2), suggesting the contribution of a host endoribonuclease. As described earlier, however, the endoribonuclease causing dmdDL seemed to be different from that involved in the 5' truncation of soc RNA. In fact, we found that this cleavage was impaired when RNase E was defective. After cells carrying the wild-type or a temperature-sensitive allele of rne were infected with T4 at a permissive (30°C) or nonpermissive (43°C) temperature, total RNAs were extracted at 2, 4, 6, 8, and 10 min after infection and analyzed by Northern blotting (Fig. 7). The soc gene can be transcribed from a middle promoter located 1.2 kb upstream as well as from its own late promoter immediately upstream. These transcripts and the 5'-truncated soc RNA are labeled P_M, F, and T, respectively, in Fig. 7. At 30°C, virtually no difference was observed between the rne alleles: transcription from the middle promoter started at 4 min and was active after 6 min, and transcription from the late promoter was detectable at 10 min. Consistent with our previous notion (8), the 5' truncation was detectable at 6 min, before late transcription started, strongly suggesting that the 5'-truncated soc RNA resulted from processing of the middle transcript. At 43°C, the 5' truncation and transcription from the late promoter in cells carrying rne⁺ started at 4 and 6 min, respectively, earlier than they did at 30°C. Again, the 5'-truncated RNA was produced before the late transcript. The temperature-sensitive rne-1 allele apparently affected transcription of soc: the level of the middle transcript was slightly low, and transcription from the late promoter was much retarded or delayed. The effect of rne-1 on transcription could be explained by reduction of the ribonucleotide pool upon T4 infection, because rne-1 impairs mRNA degradation at a nonpermissive temperature (12). The 5'-truncated RNA was not detected in cells carrying rne-1 until 8 min, and it was detectable only at a very low level at 10 min. The 5' truncation of soc RNA from plasmid-borne soc was much reduced in cells carrying rne-1 when they were cultured at a nonpermissive temperature, in comparison with that in cells carrying rne⁺ (data not shown). These results suggest that the truncation of soc RNA is attributable to RNase E.

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FIG. 7. Effects of rne on soc RNA. GW10 (rne+) and GW20 (rne-1) cells were grown at 30°C. With or without shifting of the cultures to 43°C for 30 min, cells were infected at time zero with wild-type phage. Total RNAs were extracted at 2, 4, 6, 8, and 10 min and were analyzed by Northern blotting. Band PM corresponds to the transcript from a middle promoter. Bands F and T correspond to the transcript from a late promoter and the 5'-truncated soc RNA, respectively.

Top Abstract Introduction Materials and Methods Results Discussion References

 
It was previously demonstrated that dmd mutant-specific cleavages are allele specific (8). In the present study, by assessment with various soc alleles, we found that uninfected cells possess an activity introducing such cleavages (Fig. 2). Furthermore, the host std-2 mutation completely stabilizes soc mRNA in the dmd mutant (Fig. 4) and entirely lacks dmd mutant-specific cleavages (Fig. 5). From these results, we conclude that an activity responsible for the dmd mutant-specific cleavages is encoded by the host. The observation that std-2 completely restored the growth of the dmd mutant (Fig. 3 and Table 3) strongly suggests that the cleavage activity causes dmdDL. std-2 maps in the vicinity of 59 min on the E. coli genomic map. Because there are many genes of unknown function and no known RNase genes except for rnc in this region, the causal gene may encode a novel RNase. This idea is emphasized by the inability of mutants of any of the five known endoribonucleases, RNases I*, III, E, G, and P, to support the growth of the dmd mutant (Table 2). Two different mechanisms are suggested for dmd mutant-specific cleavages. One depends on translation and cleaves at TC1, TC2, and NE of soc RNA. The other, which cleaves at TU, is independent of translation (8) (Fig. 2). Because std-2 eliminated all of these cleavages, it is likely that the gene responsible for std-2 is essential for both mechanisms.

Conjugation and transduction experiments suggest that the std-2 and std-5 loci are close to each other and distant from the std-3 locus. The suppressive effects of std-1, std-3, and std-4 on the growth defect of the dmd mutant required iscR (Fig. 6). When cloned in a multicopy plasmid, iscR can suppress the growth defect of the dmd mutant, suggesting that an increase in intracellular IscR protein levels counteracts the rapid degradation of late-gene mRNA. Based on this observation, it might be suggested that the std-1, std-3, and std-4 mutations up-regulate iscR. IscR functions as a transcriptional repressor of the iscRSUA operon, which codes for the Fe-S cluster assembly proteins IscS, IscU, and IscA (19). Accordingly, increasing IscR would reduce the amounts of the Fe-S cluster assembly proteins. Then the effects of cloned iscR on a dmd mutant could be manifested via reduced production of the Fe-S cluster proteins. Alternatively, IscR may have a novel function in addition to acting as a repressor. The latter possibility seems more likely, because the growth of a dmd mutant was still defective even in the absence of the Fe-S cluster assembly proteins, and cloned iscR also suppressed the growth defect in this case. In addition, we found that IscR can bind to RNA in vitro (N. Katayama and T. Yonesaki, unpublished data). In contrast, Dmd does not bind to RNA. Therefore, the mechanisms for suppression of RNA degradation by Dmd and IscR would be different (Fig. 8).

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FIG. 8. Model for suppression of RNA degradation by Dmd and IscR.

Our result for T4 growth on an RNase E mutant is somewhat different from the result reported by Mudd et al. (17). In their experiment, the number of wild-type progeny per infected cell was significantly reduced (50%) by the rne3071 mutation. This mutation changes an amino acid in the catalytic domain of RNase E, impairing the enzyme activity for rRNA processing as well as mRNA degradation. We used the rne-131 mutant, in which the C-terminal half of RNase E is removed, impairing mRNA degradation but not rRNA processing (14). rne-131 did not affect the growth of T4 (Table 2). These differences suggest that the C-terminal half of RNase E, which does not carry the catalytic activity but is required for efficient cleavage of a subset of RNase E substrates, is not required for T4 development.

Finally, our result suggests that the 5' truncation of soc RNA, which occurs regardless of a dmd mutation, is attributable to RNase E (Fig. 7). RNase E preferentially cleaves 5' to the AU dinucleotide in an AU-rich context followed by a stem-loop (16). The cleavage of 5'-truncating soc RNA occurs in the middle of AAAACAUUUG, but this sequence is apparently not followed by a stem-loop. A similar situation is found for an RNase E cleavage at site D of ompA mRNA (24). The 5' truncation of soc RNA normally occurs in cells carrying rne131 as efficiently as in cells carrying rne⁺ (data not shown), suggesting that the C-terminal half of RNase E is not required for the truncation.

 
We cordially thank John W. Drake at the National Institute of Environmental Health Sciences for invaluable help with the manuscript. We thank all the staff of the Radioisotope Research Center at Toyonaka, Osaka University, for facilitating our research; all of our experiments using radioisotopes were carried out at the center. We are thankful to S. Altman, S. Hiraga, H. Inokuchi, D. Kennell, S. Kushner, Y. Takahashi, and M. Wachi for providing materials.

 
* Corresponding author. Mailing address: Department of Biology, Graduate School of Science, Osaka University, 1-16 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan. Phone: 81-6-6850-5813. Fax: 81-6-6850-5817. E-mail: yonesaki{at}bio.sci.osaka-u.ac.jp.

Present address: R&D Laboratories, Nippon Organon K.K., Osaka, Japan.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, February 2003, p. 983-990, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.983-990.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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