Keywords: mRNA decay, rRNA processing, RNase P, RNase G, RNase T

Isolation and characterization of rneΔ645, a temperature-resistant revertant of the rneΔ610 allele

Because of our interest in mRNA decay, we constructed anrng::cat rneΔ610 double mutant (SK9982). When this strain was replica-plated to 44°C, a small number of temperature-resistant revertants were identified. Of eight that were tested, one (SK9987) arose from an alteration in the plasmid carrying therneΔ610 allele. DNA sequencing of the completerne gene in pMOK21 revealed a single nucleotide change, a C → A transversion within the codon for serine 418, resulting in a stop codon (Fig.1). This new premature stop codon further shortened the protein from the RneΔ610 parent by 35 amino acids, generating a polypeptide, RneΔ645, of 417 amino acids that migrated as an ∼46-kD protein in an 8% SDS polyacrylamide gel (Figs.1,2). The autoregulation of RNase E synthesis, a phenomenon first described byJain and Belasco (1995), was completely lost in therneΔ645 strain (Fig.2, lane 4). A similar loss of autoregulation has been observed previously with therneΔ610 allele (Fig.2, lane 3;Ow et al. 2000). To confirm that the C → A transversion was responsible for the suppression of lethality observed in SK9987, we used site-directed mutagenesis to reconstruct therneΔ645 mutation in pMOK15 (rneΔ610Cm^r), creating pMOK29 (rneΔ645 Cm^r). Transformation of pMOK29 into SK9705 and the subsequent displacement of the resident plasmid (pQLK26,rne⁺ Km^r) resulted in a strain, SK10103, whose properties were identical to SK9987.

Growth properties of the rneΔ645 mutant

On Luria agar medium at 44°C, SK9987 (rneΔ645) took 2 d to form large colonies compared with 1 d for therne⁺ control (SK9714). In Luria broth, SK9987 continued to grow at 44°C (35 min generation time), albeit more slowly than the wild-type SK9714 control (26 min; Fig.3A). In contrast, SK9957, therneΔ610 progenitor, ceased growing at ∼4 h after the shift to the nonpermissive temperature (Fig.3A). A control strain, SK9937, harboring therne-1 temperature-sensitive allele ceased growing between 60 and 90 min after the shift to 44°C (Fig.3A).

Because these strains contained low-copy-number plasmids (6–8 copies/cell) carrying therne⁺,rne-1,rneΔ610, andrneΔ645 alleles, we also determined the growth properties of cells carrying these mutations in a single copy. To accomplish this, therne⁺,rne-1,rneΔ610, andrneΔ645 alleles were moved into pMOK40 (Ow et al. 2002), a single-copy vector carrying Sm^r/Sp^r. Under these conditions, both therne-1andrneΔ610strains ceased growing at ∼120 min after the shift to 44°C, whereas therneΔ645 andrne⁺ strains continued growing (Fig.3B). Interestingly, the increased copy number of therne-1 allele caused a more rapid cessation of cell growth (cf. SK9937 and SK10144 in Fig.3A,B). In contrast, the generation times for therneΔ645strains at 44°C were 35 min (SK9987, 6–8 copies) versus 47 min (SK2685, single-copy). Based on Western blot analysis, there was a three-to fourfold decrease in the amount of the RneΔ610 and RneΔ645 proteins when the mutations were present in the single-copy plasmid compared with the 6–8-copy-number plasmid (data not shown).

mRNA decay properties of the rneΔ645 mutant

Previous analysis of therneΔ610 allele had suggested that the conditional lethality associated with therne-1andrne-3071mutations was not related to defects in mRNA decay (Ow et al. 2000). To determine if this hypothesis were correct, we examined the decay of two RNase E-dependent transcripts,rpsT andrpsO (Mackie 1991;Hajnsdorf et al. 1994) at both 37°C and 44°C in therneΔ645 mutant. At 37°C, both therpsO andrpsT mRNAs decayed between 1.5- and 1.7-fold more rapidly in SK9987 (rneΔ645) than in SK9957 (rneΔ610), but their decay rates were still much slower than those obtained inrne⁺ andrne-1strains (Table1A). In fact, the half-lives in therneΔ645 strain at 37°C were still equal to or longer than those observed in anrne-1 mutant at the nonpermissive temperature (Ow et al. 2000).

We also determined the half-lives of therpsO, rpsT, andpnp transcripts at 44°C in strains carrying single copies of therneΔ610andrneΔ645 alleles (Table1B). Under these conditions, mRNA decay in therneΔ645 strain was either more defective than in therne-1 mutant (rpsO) or not significantly better (rpsT andpnp; Table1B). It should also be noted that with all three transcripts, the half-lives increased considerably in therneΔ610 strain relative torne-1, even though therne-1 strain ceased growing sooner at 44°C (Fig.3B). In contrast, the half-lives of all three transcripts in therneΔ645mutant were comparable to those observed in therne-1 strain even though it continued growing at 44°C (Fig.3B; Table1).

Comparison of 9S rRNA processing in the rneΔ610 and rneΔ645 mutants

Because we had previously observed that 9S rRNA processing was relatively normal in therneΔ610mutant (Ow et al. 2000), we did not expect to see a significant difference between therneΔ610andrneΔ645strains. To confirm this prediction, we probed a Northern blot containing total RNA, isolated fromrne⁺,rne-1, rneΔ610, andrneΔ645 strains that had either been grown at 37°C or had been shifted to 44°C for 150 min, with an oligonucleotide complementary to 5S rRNA. As expected, in therne-1 strain, larger rRNA precursors accumulated when the strain was shifted from 37°C to 44°C (Fig.4). In contrast, there were significantly fewer precursors in both therneΔ610 andrneΔ645 mutants at 44°C (Fig.4). In fact, the steady-state levels of mature 5S rRNA were identical in the wild-type,rneΔ610, andrneΔ645 strains at 44°C compared with a 32% reduction in therne-1 mutant (Fig.4).

Comparison of M1p processing in the rneΔ610 and rneΔ645 mutants

It has been shown that RNase E is involved in the maturation of the M1 RNA subunit of RNase P (Gurevitz et al. 1983;Lundberg and Altman 1995). Because this enzyme is required for the generation of the mature 5′ ends of tRNAs (Altman et al. 1995), we determined the half-life of the M1 precursor (M1p) in therneΔ610andrneΔ645strains to ascertain whether a change in the processing of this transcript could account for the difference in cell growth at 44°C (Fig.3A,B). In therne⁺ control, processing occurred too rapidly to determine a half-life (Table2). In contrast, when therne-1,rneΔ610, andrneΔ645alleles were present in 6–8 copies/cell, the half-life of the M1p was identical within experimental error (Table2). When therne alleles were present in single copy, the M1p half-life in therne-1andrneΔ645 strains was almost identical, whereas it was almost four times longer in therneΔ610mutant (Table2).

Processing of polycistronic transcripts containing tRNAs and tRNAs encoded within rRNA operons requires functional RNase E activity

Because the changes in mRNA decay rates as well as 9S rRNA and M1 processing did not seem sufficient to account for the ability of therneΔ645strain to grow at 44°C, we next analyzed the processing of tRNAs inrne⁺,rne-1,rneΔ610, andrneΔ645 strains at 44°C. If RNase E were required for tRNA processing, we assumed that its most likely role would be in the cleavage of polycistronic operons that contained several tRNAs. Initially, we chose to analyze the glyW cysT leuZ (Fig.5A) and argX hisR leuT proM polycistronic transcripts (data not shown) because the cysT and hisRgenes are unique.

Using an oligonucleotide probe (cysT–leuZ), we carried out a half-life experiment for the intercistronic region between thecysT andleuZ genes (Fig.5A,B). Although no precursor transcripts were visible in the wild-type control, two large species of 460 (full length) and 290 nucleotides (nt) were observed in therne-1,rneΔ610, andrneΔ645 mutants, along with a series of smaller intermediates ranging in size from 90 to 200 nt (Fig.5B). Half-life determinations for the largest species showed that therneΔ645 strain processed it twofold faster than therneΔ610 mutant and 2.8-fold faster than therne-1strain (Table3; Fig.3D). As a control, the membrane was stripped and reprobed for the mature tRNA^Cys (Fig.5D).

To confirm that these observations were not unique to theglyW cysT leuZ operon, we next tested theargX hisR leuT proM polycistronic transcript. A probe (hisR–leuT) for the spacer region betweenhisRand leuT detected two large precursor transcripts of 530 (full length) and 335 nt in addition to smaller species between 85 and 225 nt in length in therne mutants (data not shown). Again, no precursors were seen in the wild-type control. With this operon, the decay of the full-length precursor was almost identical in therne-1 andrneΔ610 strains (Table3; Fig.3D) and much slower than that observed for theglyW cysT leuZ operon. However, the processing in therneΔ645 strain was 3.7-fold faster than in either therne-1 orrneΔ610 mutants (Table3; Fig.3D).

We also examined whether RNase E was involved in the processing of tRNA operons that contained multiple copies of specific tRNAs. For these experiments we chose thetyrT tyrV tpr and lysT valT lysW valZ lysY lysZ lysQ operons. In both cases, significant amounts of processing intermediates were observed in the threerne mutants compared with none in the wild-type control (data not shown). Similar differences in tRNA processing were observed when therneΔ610andrneΔ645 alleles were present in single copy (data not shown). As a final experiment, we tested the fourglt tRNAs because they are all contained within rRNA operons (rrnB, rrnC, rrnE, andrrnB). As was seen with polycistronic tRNA transcripts, functional RNase E activity was required to obtain mature tRNA^Glt (data not shown).

Processing of monocistronic tRNA transcripts also requires functional RNase E activity

Because monocistronic transcripts have relatively short 3′ ends, we assumed that the multiple 3′ → 5′ exonucleases (RNase II, RNase BN, RNase D, RNase PH, RNase T, and PNPase) that have been shown to be involved in the generation of mature 3′ CCA termini (Li and Deutscher 1996) would be sufficient for maturation. To determine if this hypothesis were correct, we examined the processing of thepheU transcript because it contains 37 nt downstream of the mature CCA terminus (Fig.6A) and it is almost identical in sequence organization topheV(contains a 36-nt downstream sequence), the other phenylalanine tRNA inE. coli. Thus, analysis of thepheU transcript allowed us to simultaneously measure processing of both phenylalanine tRNAs.

To our surprise, the 116–117-nt-long primary transcript of tRNA^Phe was observed in all threernemutant strains (Fig.6B). Of even more interest were the longer half-lives for thepheU/pheV precursors compared with the polycistronic transcripts (e.g., Table3, forrne-1 cf. the half-lives of 31.8 min for thepheU andpheV precursors with 9.9 min forcysT leuZ and 14.5 min forhisR leuT). However, as was seen for the polycistronic operons, processing was significantly faster in therneΔ645 strain compared with either therne-1 orrneΔ610 mutants (Table3; Fig.3D). No intermediates were detected in therne⁺ strain (Fig.6B). When these experiments were repeated with therneΔ610 andrneΔ645 alleles in single copy, the 116–117-ntpheU/pheV precursors were processed fivefold more rapidly in therneΔ645 strain compared with therneΔ610mutant (data not shown).

To show thatpheU andpheV were not unique among the monocistronic tRNA transcripts, we also examined the maturation of tRNA^Asn under steady-state conditions. There are four copies of this gene in theE. coli genome (asnT,asnU, asnV, andasnW), each transcribed as a monocistronic transcript of a different length. Inactivation of RNase E led to a large increase in the amount of the four precursor tRNAs (see Fig.9, below).

When we compared the differences in tRNA processing rates (Fig.3D) with the variations in the mRNA decay half-lives (Fig.3C) and the growth profiles at 44°C associated with therne-1,rneΔ610, andrneΔ645 strains (Fig.3A,B), we observed a direct correlation between cell growth and tRNA-processing efficiency.

Maturation of the 5′ terminus of tRNA precursors requires prior RNase E cleavage

To determine if RNase E cleavage was the rate-limiting step in tRNA maturation, we performed two different types of experiments. Previous in vitro experiments byRay and Apirion (1981a) suggested that the action of RNase E could influence RNase P cleavage at the 5′ terminus of an unprocessed tRNA transcript. Accordingly, to examine whether in vivo RNase P processing of the 5′ terminus was affected by RNase E cleavage at the 3′ end, we performed Northern analyses with probes designed to detect the presence of unprocessed 5′ termini. First, we tested for 5′-end processing of thetyrTtRNA, which is the first gene in thetyrT tyrV tpr transcript and contains a 44-nt 5′-terminal region (Fig.7A). Although no processing intermediates were observed in the wild-type control, a variety of larger species were observed in the threernemutants, indicating that RNase P cleavage required prior activity by RNase E at the 3′ end (Fig.7B).

Although this result strongly suggested that RNase P did not process the 5′ end of thetyrTprecursor prior to RNase E cleavage at the 3′ end, we wanted to confirm this observation using a simpler substrate. Accordingly, we examined the steady-state levels of six monocistronic transcripts (asnT, asnU, asnV,ansW, pheU, andpheV) in wild-type,rne-1, rnpA49, andrne-1 rnpA49strains after a 150-min shift to 44°C. In the case of tRNA^Asn, the four unprocessed monocistronic transcripts are 230 nt (asnT), 210 nt (asnW), and 120 nt (asnU andasnV) in length (Figs.8A,9). Each transcript contains ∼9–10 nt upstream of the mature 5′ end. In anrnpA49 mutant, which encodes a temperature-sensitive mutation in the protein subunit of RNase P (Apirion and Gitelman 1980), there was a large increase in an 85-nt species that represented all four tRNA^Asn species that had been processed by RNase E at their 3′ ends but not by RNase P at their 5′ termini (Fig.8A). In contrast, in therne-1 strain, the full-length precursors (230, 210, and 120 nt) accumulated. If RNase P functioned prior to RNase E cleavage, one would have expected to see species of ∼220, ∼200, and ∼110 nt in therne-1 mutant. The fact that the tRNA^Asn band profile in thernpA49 rne-1 double mutant was identical to those seen in the two single mutants further suggested that all potential processing intermediates were accounted for.

A similar result was obtained with thepheUand pheV transcripts (Fig.8A). In this case, the full-length transcripts are 116 nt (pheV) and 117 nt (pheU), respectively, and contain a very short 5′ leader (3–4 nt). In thernpA49 mutant, two species (80–81 nt) differing in length by 1 nt accumulated, containing the immature 5′ end and 1–2 extra nucleotides at the 3′ end. In contrast, in therne-1mutant, only the full-length transcripts accumulated. Again, there was no evidence for an intermediate that contained a mature 5′ end and an immature 3′ end.

Although the steady-state analysis indicated that RNase P processing of tRNA precursors did not occur prior to RNase E cleavage, we also performed kinetic experiments with both tRNA^Asn (Fig.8B) and tRNA^Phe (data not shown) following a shift to 44°C. At the time of the shift, the mature tRNA^Asn was the major species in therne⁺ andrne-1 strains (Fig.8B). In thernpA49mutant there was also a low level of the 85-nt product, indicating that RNase P activity was slightly defective at 30°C. In thernpA49 rne-1 double mutant, there were higher levels of all the precursors. More importantly, in therne-1strain the four full-length tRNA^Asn transcripts accumulated over time, whereas the 85-nt species containing the unprocessed 5′ ends rapidly appeared in thernpA49 strain. With thernpA49 rne-1 double mutant, all of the precursors accumulated following the shift to 44°C (Fig.8B). The kinetic analysis confirmed the results obtained with the steady-state experiment (Fig.8A).

Analysis of tRNA processing efficiency at 44°C

In our Northern analysis of various tRNA transcripts, we noticed considerable differences in the half-lives among the various tRNA precursors (Table3), suggesting a possible hierarchy of substrates for RNase E, whereby some were cleaved more efficiently than others. Accordingly, we examined the steady-state levels of 59 tRNA transcripts in therne⁺,rne-1, rneΔ610, andrneΔ645 strains at 44°C under conditions such that each allele was present in 6–8 copies/cell. These experiments showed that there were three classes of tRNA substrates for RNase E. One group (Class 1) including tRNA^Asn (Fig.9), tRNA^Val, tRNA^Tyr, tRNA^Gly, tRNA^Phe, and tRNA^Met were very efficiently processed in therne⁺ strain such that between ∼95% and ∼99% of the tRNA was present in the mature form (PF) under steady-state conditions. At the other extreme (Class 3), tRNA^His (Fig.9), tRNA^Cys, and tRNA^Arg were inefficiently processed such that only ∼28%–30% of the transcripts were mature in the wild-type strain under identical conditions. Class 2, represented by tRNA^Pro (Fig.9), tRNA^Glt, and tRNA^Leu, had PF values that ranged between 75% and 85%. Similar differences in PF values were observed when thernealleles were present in single copy (data not shown).

Significant differences among the PF values for the various tRNAs were also observed in therne-1, rneΔ610, andrneΔ645 mutants (Fig.9; data not shown). For example, the PF in therne-1 strain was always lower than that obtained in therne⁺ control. In the case of tRNA^His, there was a 14-fold reduction, whereas with tRNA^Pro the decrease was 2.8-fold (Fig.9). In addition, the PF was always greater in therneΔ645strain compared with therne-1 andrneΔ610mutants (Fig.9; data not shown). Furthermore, irrespective of their relative processing efficiencies, the steady-state amounts (RQ) of each tRNA were alwaysrne⁺ > rneΔ645 > rneΔ610 ≥ rne-1. This relationship correlated well with the growth profiles of these strains at 44°C (Fig.3A,B). These data were also in complete agreement with the relative tRNA precursor half-lives described in Figure3D.

Mapping RNase E cleavage sites within tRNA precursors

Because RNase E appeared to play an integral role in the processing of all the 59 tRNA transcripts we tested, we carried out a series of primer extension experiments to map some of the actual cleavage sites. When we tested thetyrT transcript, there was a single cleavage site located 1 nt downstream of the mature CCA terminus (CCAU↓AAUUCACC; Table4; Fig.10). This result was confirmed by performing a Northern blot fortyrTusing steady-state RNA isolated from aΔrnt::kan rph-1 strain (SK10148), which should not be able to generate a mature 3′ end because of the loss of RNase T and RNase PH (Li and Deutscher 1994,1996). No large precursors were observed, but an ∼86-nt species, 1 nt longer than the mature tRNA^Tyr, was present in significant amounts (data not shown). In a second experiment, we determined the RNase E cleavage site between thelysY andlysZgenes. In this case, cleavage also occurred 1 nt downstream of the mature CCA terminus (CCAG↓UUUUA ACA; Table4).

Because both of the RNase E cleavage sites mapped very close to the mature CCA terminus and fell within an AU-rich region, we decided to compare the downstream sequences of the three classes of tRNAs identified by the experiments described in Figure9. In particular, we wanted to see if there were any correlations between those tRNAs that were processed inefficiently (his, cys, andarg) and those that were processed efficiently (asn,val, tyr, gly, andmet). As shown in Table4, most of the 3′ regions are AU-rich. In general, in the efficiently processed tRNAs there is usually an A residue immediately downstream of the CCA, but in those inefficiently processed there is either a C or a U (Table4). However, the significance of this observation is called into question by the fact that some of the intermediately processed tRNAs likeleuVhave a 3′-terminal region that is almost identical to that ofmetV andmetZ (Table4). In addition, there are a number of 3′ ends such asvalZ that appear to be processed efficiently but are GC-rich (Table4).

Bacterial strains and plasmid displacement

TheE. coli strains used in this study are listed in Table5. Strains SK2525, SK2534, SK2681, SK2685, SK9982, SK9987, SK10103, SK10143, SK10144, and SK10148 were constructed by a combination of P1 transduction and plasmid displacement (Ow et al. 2000,2002). Details of these constructions are available upon request.

Plasmid construction

The plasmids used are listed in Table5. Vector pMOK18 was made by inserting anSmaI/SmaI streptomycin/spectinomycin-resistance cassette from pKRP13 (Reece and Phillips 1995) into theScaI site of pWSK29 (Wang and Kushner 1991). To make pMOK19 (rneΔ610 Sm^r), aKpnI/SacI DNA fragment from pMOK15 (rneΔ610 Cm^r;Ow et al. 2000) containing therneΔ610 allele was inserted into theKpnI/SacI sites of pMOK18. pMOK21 (rneΔ645 Sm^r) was isolated naturally from a 44°C replica plate of SK9982. pMOK29 (rneΔ645 Cm^r) was made using site-directed mutagenesis using pMOK15 as the template. Plasmids pDHK2 and pMOK45 were constructed by digesting pMOK15 (rneΔ610) and pMOK13 (rne-1), respectively, withSacI andKpnI and ligating the DNA fragment containing therne mutant allele into aSacI/KpnI-digested pMOK40 (Sp^r) vector (Ow et al. 2002). pDHK6 was made by inserting anEcoRI/NotI fragment carrying therneΔ645 allele into theEcoRI/NotI sites in pMOK40.

Growth curves

Cultures were routinely grown in Luria broth with thymine (50 μg/mL) and chloramphenicol (20 μg/mL), kanamycin (50 μg/mL), spectinomycin (20 μg/mL), or streptomycin (20 μg/mL) at 37°C or 30°C with agitation until they reached 40–45 Klett units (1 × 10⁸–1.1 × 10⁸ cells/mL; No. 42 green filter). Once reaching this density, cultures were shifted to 44°C. Every 30 min thereafter, cells were diluted with fresh prewarmed medium. Klett values were adjusted with the appropriate dilution factor.

DNA sequencing

Sequencing of therneΔ645 allele was done using an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems) as instructed by the manufacturer using primers RNE+240, RNE+292, RNE+747, RNE+1006, and RNE+3628. These primers encompassed therneΔ610 andrneΔ645 genes in their entirety.

Site-directed mutagenesis

The introduction of a stop codon into plasmid pMOK15 (rneΔ610) to generate therneΔ645 allele (pMOK29) was done with the U.S.E. Mutagenesis Kit (Amersham Pharmacia) following the instructions of the manufacturer. The selection and the mutagenesis primers (which convert the codon for serine 418 from TCG to a TAG stop codon) were RNE-893 and RNE+1593T, respectively. DNA sequencing at the site of the C → A transversion and RNase E Western blot analysis on strain SK10103 were performed to verify the authenticity of the mutation.

Western analysis

Western analysis was performed as described previously (Ow et al. 2000,2002).

Primer extension analysis

Primer extensions were carried out using Thermoscript RNase H⁻ reverse transcriptase (Invitrogen) at 60°C according to the manufacturer's specifications. The sequences for the various primers used in the hybridization reactions are available upon request.

Northern analysis

For RNA isolation and Northern analysis, we followed the procedures described inO'Hara et al. (1995) andBurnett (1997). All of the RNAs used for the tRNA-processing Northern blots were obtained from 44°C cultures diluted as described for the growth curves so the cultures were maintained in exponential growth at all times. RNA was obtained from exponentially growing cultures that had been shifted to 44°C for 2 h (SK9937) and 4 h (SK9714, SK9957, and SK9987). These times were chosen based on the times after shift at which therne-1andrneΔ610strains stopped growing (Fig.3A). The 9S rRNA Northern was done using RNA extracted from cultures shifted to 44°C for 150 min. For strains carrying single copies ofrne-1,rneΔ610, andrneΔ645and thernpA49andrnpA49 rne-1 mutants, RNA was isolated after a 150-min shift to 44°C. RNAs from MG1693 (rph-1) and SK10148 (Δrnt::kan rph-1) were harvested from exponentially growing cultures at 37°C. Band densities were quantitated with a Phosphorimager series 400 (Molecular Dynamics). Decay rates for half-life determinations were calculated using linear regression analysis.

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