Site-specific endonucleolytic cleavages and the regulation of stability of E. coli ompA mRNA

Cell, Vol 52, 893-901, March 25. 1988, Copyright (ci 1988 by Cell Press

Site-Specific Endonucleolytic Cleavages and the Regulation of Stability of E. coli ompA mRNA

ajar Melefors and Alexander von Gabain Institute for Applied Cell and Molecular Biology Umea University S-901 87 Umea, Sweden and Department of Bacteriology Karolinska Institute S-104 01 Stockholm, Sweden*

The stability of ompA mRNA is growth-rate dependent. We show that the 5’ noncoding region of this mRNA provides a target for site-specific endonucleases. The rate of degradation of ompA mRNA parallels the rate of these endonucleolytic cleavages, implying that endonucleolytic rather than exonucleolytic attack is the initial step in ompA mRNA degradation. Thus the 5’ noncoding region appears to be a determinant of mRNA stability, and endonucleolytic cleavages in the 5’ noncoding region may well regulate expression of the ompA gene.

Introduction

The level of gene expression is determined primarily by three factors: the rate of transcription, the efficiency of translation, and the stability of the messenger RNA. The basic factors influencing transcription and translation have been identified and intensively studied, whereas the regulation of mRNA turnover is poorly understood (for re- view, see Higginsand Smith, 1986). In Escherichiacoli the stability of different mRNA species can vary by at least a factor of 50 (Pedersen et al., 1978; Nilsson et al., 1984). The half-lives of some E. coli mRNA species are growth- rate dependent (Nilsson et al., 1984). One mRNA species under growth-rate control is the monocistronic ompA message, encoding the major outer membrane protein OmpA. Furthermore, the rate of OmpA protein synthesis correlates with the growth-rate dependent changes in half-life of ompA mRNA.

In eukaryotic cells mRNA stability can be regulated by external stimuli, e.g., hormones (Brock and Shapiro, 1983). Deficiencies in mRNA degradation are associated with clinical conditions such as certain forms of thalassemia or Burkitt’s lymphomas; in these cases the 8-globin mRNA and the myc mRNA, respectively, exhibit abnormal stability (Maquat et al., 1981; Eick et al., 1985).

In E. coli some mono- and polycistronic transcripts decay at different rates for different segments (von Gabain et al., 1983; Belasco et al., 1985; Newbury et al., 1987). Thus it has been suggested that the decay of a transcript, or a segment thereof, is controlled by a rate-limiting step followed by rapid degradation (Blundell and Kennell, 1974;

’ Present address

von Gabain et al., 1983; Schmeissner et al., 1984; Belasco et al., 1985).

The 5’ noncoding region of mRNA has been suggested as a possible target for such a rate-limiting event, since these sequences apparently control the stability of certain mRNA species (Gorski et al., 1985; Belasco et al., 1986). Endonucleolytic cleavages could provide the initial step in mRNA degradation. However, as yet there is no direct evidence that the endonucleolytic cleavage provides the rate-limiting step in mRNA degradation (Higgins and Smith, 1986). The present study identifies site-specific cleavages in the 5’ noncoding region of the ompA mRNA that appear to be the initial step in degradation and may therefore control ompA mRNA turnover.

Results

Decay of a Hybrid tat-ompA Transcript The detection of mRNA cleavage products should be facilitated by transient overproduction of the message. Such overproduction can be achieved using plasmid pTac- ompA, in which ompA transcription is directed from the isopropyl 8-o-thiogalactoside (IPTG)-inducible tat promoter (see Experimental Procedures). The ompA gene fused to the tat promoter includes the transcription terminator but not the promoter (Movva et al., 1981; von Gabain et al., 1983). The transcript initiated at the tat promoter is 54 nucleotides longer than the wild type ompA transcript.

pTac-ompA was transformed into E. coli strain BRE50 (which does not express the chromosomal ompA gene), and decay of the tat-ompA transcript (see Figure 1) was analyzed after IPTG induction. Analysis of induced RNA by Northern blotting showed that, as expected, the amount of ompA-specific transcript increased after IPTG induction and decreased after transcription was blocked with rifampicin. No ompA-specific transcripts were detect- able before IPTG induction; after induction the amount of tat-ompA transcripts was about 6 times higher than the amount of ompA mRNA in wild type cells (Figure 2, Table 1). In addition to the full-length tat-ompA mRNA (1269 nucleotides), an mRNA species loo-150 nucleotides smaller could also be distinguished (Figure 2). Two larger mRNA species were also observed, which presumably reflect transcripts that terminate downstream of the major termi- nation site.

Apart from the smaller, 1119-1169 nucleotide mRNA species described above, no mRNA species were found that were shorter than the full-length mRNA species (Fig- ure 2). All mRNA species detected with the ompA probe appeared after IPTG induction, excluding the possibility that the various transcripts arise from multiple promoters. When we analyzed the ompA mRNA from a wild type C600 strain, we were unable to detect the smaller, 1119-1169 nucleotide mRNAs without using more sensr- tive techniques (see below).

The increase in ompA mRNA synthesis after IPTG induction might be expected to result in a higher level of ma-

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tat-ompA A B C 0 E

200 nt Figure 1 Physlcal Map of the tat-ompA Transcript

The wavy hne lndlcates the 5’ noncodmg region, the solid bar defines the translated region, and the triangle marks the site of fusion between the

rat and the wild type ompA sequences The map IS truncated at the 3’ end Thin lines underneath show the DNA probes used in these studies Closed circles and restrIctIon sites, EcoRl and Avall, correspond to the ends of the probes that were labeled. The posItIons of the 5’ and 3’ ends

of the Sl-resIstant DNA fragments were located wlthln the sequences lndlcated by bars A, 8, C. D, and E lndtcate the 5’ ends of the Si-resistant DNA fragments ldentlfled using the ‘AvaIl“ probe (“downstream” products) y, w, v, and u Indicate the 3’ ends of the Sl-reslstant DNA fragments Identi- fied using the “EcoRI“ probe (“upstream” products) The primer used for the primer extension was ldentlcal at Its 5’ end to the ‘Avall” probe

ture OmpA protein. Cellular extracts were prepared from plementary to the 5’ region of the tat-ompA transcript

IPTG-induced and from noninduced cultures and were Figure 1). Sl analysis identified the full-length transcript *

fractionated by SDS-polyacrylamide gel electrophoresis initrating at the tat promoter (Figure 4) (Bremer et al.,

(Figure 3). The results show that the level of mature OmpA 1980; Fiirste et al., 1986). In addition, five shorter tran-

protein is significantly higher when cells are induced with scripts (A-E) were identified, reflecting multiple 5’ end-

IPTG. points (Figure 4).

Specific Endonuclease Cleavage in the 5’ Region of tat-ompA mRNA To elucidate the origin of the smaller, 1119-1169 nucleotide mRNA species, we used Sl nuclease mapping to identify the 5’ endpoints. RNA samples were probed with a 5’ end-labeled DNA probe (the “Avall” probe) com-

ompA IPTG RIF w’ 1 I

23S-

16S-

1215- - 4-

The mRNA 5’ termini corresponding to DNA fragments f3, C, D, and E reside in the 5’ noncoding region of the ompA transcript (Figure 1). The mRNA terminus reflected by DNA fragment A maps within the 54 additional 5’-terminal nucleotides of the hybrid transcript, which are absent from the native ompA mRNA (see above). In addition, one major and several minor protected DNA fragments showed

ht) Figure 2 Northern Blot Analysis of Over- productlon and Decay of fat-ompA mRNA

(Left) Total cellular RNA was Isolated from E co11 BRE50 cells (no chromosomal ompA ex~ presslon) harboring pTac-ompA, at appropriate time points before and after InductIon of tran- scrIptIon by IPTG (-15 mln) and after transcnp- tlon was blocked with rlfamplcln (RIF) (0 mln) Ten mlcrograms of total RNA was loaded Into each lane The upper arrow at right lndlcates the full-length transcript. and the lower arrow

1353-

1078-

- Indicates the smaller mRNA species The first

w lane shows wild type ompA mRNA (10 rig of total RNA from C600 cells) The size of the wild type VanscrIpt was taken from a previous publl- catton and IS lndlcated in nucleotldes (van Gabaln et al 1983) The sizes of the tac~ompA mRNA species were estimated using the wild type ompA mRNA and the 23s and 16s rRNAs as size markers (Right) For a better sue determlnatlon the tac- ompA mRNA (arrows at right) was fractionated alongsIde Haelll-digested qX174 DNA (sizes are lndlcated at left in nucleotldes) The expert- merit also shows the absence of tat-ompA transcripts smaller than the 1119-l 169 nucleotlde

mRNA species Both Northern blots were probed with ntck-

translated pTU500 DNA

Regulation of E. co11 ompA mRNA Stabrlrty 895

Table 1. Stability of Weld Type ompA and tat-ompA mRNAs rn E. co11 under Drfferent Growth Condrtrons

Relative Amount of Ratio of Cleavage Full-Length ompA Product D to Full-Length

Transcript Half-Life (m(n) mRNA per Total RNA Transcrrpt

Wild type ompA (L broth) 25 + 2 10 0.002 Wild type ompA (MOPS-succrnate) 7&l 0.35 0 006 tat-ompA before IPTG (L broth) <2’ 0.4 0.012 tat-ompA after IPTG (L broth) 2.25 t 0.25 63 0.075 tat-ompA after IPTG (MOPS-succtnate) 1 25 f 0 25 N.D. 0 246

mRNA half-ltves were dewed from the experiments shown rn Figure 8. The half-life of lac-ompA mRNA before IPTG Inductton (‘) was estimated to be shorter than 2 mm the small number of transcrrpts (corresponding to the 295 nucleotrde DNA segment obtained by prrmer extensron) made it drfftcult to establish an accurate half-life. However, 2 mtn after the addrtron of rrfampicm more than half of the full-length transcripts were degraded (data not shown).

The relative amounts of full-length ompA mRNA, per total RNA, were deduced from the experrments shown tn Figures 2, 4, and 7. Relative amounts of weld-type ompA mRNA and fat-ompA mRNA were dewed from the data rn Figure 2, relatrve amounts of fat-ompA mRNA before and after IPTG Inductton were dewed from the data rn Figure 4; and the relative amount of weld-type ompA mRNA at dtfferent growth rates was from Figure 76. The amount of weld-type ompA mRNA (L broth) per total RNA was arbitrartty set at 1 0, all other values were normalrred to thus value N.D , not determined.

The ratios of cleavage product D to full-length transcrrpt were obtained from the experiments shown tn Figures 4 and 7. The ratios observed rn independent experiments (e.g., Figures 7A and 76) drd not deviate stgnrftcantly from one another

the presence of transcripts that have 5’ ends within the ompA coding region. The 5’ end of the transcript defined by DNA fragment E maps only about 15 nucleotides upstream of the AUG start codon of the ompA transcript, close to the predicted ribosome binding site (Shine and Dalgarno, 1974) (Figure 1).

Since the shorter mRNA species are not a result of extra promoters, we can best explain them as processing products of the full-length transcript. We noted that the increased sensitivity of Sl analysis, in contrast to Northern

I PTG

COmpA

a -

Frgure 3. SDS-Polyacrylamrde Gel Electrophorests of the OmpA Pro- tein before and after IPTG Inductron

E. colt GNlO cells (no chromosomal ompA expression) harboring plasmrd pTac-ompA were grown to exponentral phase, and half of the culture was Induced wrth IPTG. The same number of cells was taken from the Induced and the nonrnduced cultures, and the total cellular protern was analyzed by 6% SDS-polyacrylamrde gel electrophoresrs The mature OmpA protern, rndrcated by an arrow at rtght. was rdentrfted by antrbody prectprtatron rn a prevrous expertment (data not shown)

blotting, made it possible to detect some tat-ompA message even in the absence of IPTG induction. This result indicates that the tat promoter, unlike the lac promoter, cannot be fully repressed by the lac repressor protein.

To confirm the above results using an independent method, primer extension was used to map the 5’ endpoints of the fat-ompA mRNA. RNA samples isolated after IPTG induction and rifampicin inhibition were annealed with a 5’ end-labeled, synthetic DNA primer and transcribed with reverse transcriptase. To obtain comparable results, the sequence of the DNA primer was identical to the 5’ end of the “Avall” probe used for Sl analysis. The transcript endpoints identified by primer extension and by Si nuclease analysis were identical in number and loca- tion (Figure 4).

Degradation of 5’-Processed Transcripts The degradation of the different mRNA species after

rifampicin additron was examined. Only the degradation of DNA fragment *, reflecting the full-length tat-ompA mRNA, showed the expected decay profile. The other DNA fragments, A-E, represent the amount of processed mRNA, which is determined both by the rate of processrng of full-length mRNA and the stability of the processed species. The amounts of transcripts A, 6, and C began to decrease immediately after the addition of rifampicin; the amount of transcript D decreased after a lag period; and the amount of transcript E increased initially before start- ing to decrease. This accumulation of DNA fragment E immediately after rifamprcin addition emphasizes the fact that the processing products are derived from the full- length mRNA. This result was investigated further by analyzing accumulation of DNA fragment E after inhibition of transcription with two different concentrations of rifampicin. Ten minutes after rifampicin addition, DNA fragment E had only accumulated in the presence of the higher concentration of rifampicin (Figure 5). However, the amount of full-length fat-ompA mRNA (DNA fragment *) was the same at both rifampicin concentrations.

Cell 896

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25 400 (Wffl)

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0 10 10 oni") Figure 5. Decay of tat-ompA mRNA at Different Concentrattons of Rifampicin

RNA samples were obtatned as descrrbed rn Figure 2 Ceils were exposed to IPTG for 15 min, and, after the “0 min” sample was taken, the culture was split and rifampicin was added at the indicated concentrations. Ten minutes later samples were taken for RNA extraction and analyzed as described rn Frgure 48. DNA fragments * and E are indt- cated as in Figure 4. DNA fragment E accumulated to htgher levels when 400 nglml rrfamprcin was used.

Figure 4. Sl Nuclease and Reverse Transcrip- tase Analysis of tat-ompA mRNA wrth the “Avall” Probe

(A) RNA samples were obtained as described rn Figure 2, and annealed with the 5’ end- labeled “AvaIl” probe (Figure 1). Hybrids were treated with Sl nuclease and analyzed by 6% denaturing polyacrylamrde gel electrophore- SIS. Radtolabeled Haelll-digested (pX174 DNA was used for size markers (at left). Trme points are indicated at which samples were collected and at which IPTG (-15 mm) and rifamprcrn (RIF) (0 mm) were added to the culture. The au- toradiograph was overexposed to detect minor bands, and hence the stronger signals have saturated the X-ray ftlm. (6) Reverse transcriptase analysis of tat-ompA mRNA. RNA samples were obtained as described In Figure 2, and cells were exposed to IPTG for 15 min. Rifampictn was added, and 0, 5, 10, 15, and 25 min thereafter RNA samples were isolated. RNA samples were annealed with the 5’end-labeled DNA oligomer (homolo- gous to the S’end of the “Avall” probe) and transcribed wtth reverse transcriptase, and the DNA products were analyzed by 6% denatur- mg polyacrylamide gel electrophoresis.

*, A, 6, C, D, and E indicate the major Si- resistant DNA fragments or primer extension products that were found in the 5’ noncodmg region. The sizes of the fragments in nucleotides are as follows: ‘, 295 ? 12; A, 244 f 10; 6, 190 2 7; C, 168 r 6; D, 143 + 6; and E, 124 + 5. *corresponds to the full-length tac- ompA mRNA.

If the many different ti’ends of ompA mRNA are a result of endonucleolytic cleavages, complementary cleavage products extending toward the 5’end of the transcript (“upstream” products) should also be identifiable. A DNA probe was made by labeling the 3’ end of the unique EcoRl site in pTac-ompA (Figure 1) and was used to search for such molecules. Sl analysis revealed such upstream cleavage products to have 3’ ends complementary to the 5’ ends of the above-identified mRNAs (“downstream” cleavage products) (Figures 1 and 6). In three cases (u and E, wand C, y and B) the 3’and S’ends of the upstream and downstream products match perfectly, allowing for experimental error in size determination. However, one case (the sizes of counterparts D and v) indicates a gap between the 3’ and 5’ ends. This may be due to the action of 3’-to-5 exonucleases (Donovan and Kushner, 1986), which are known to trim or completely degrade RNAs with unprotected 3’ ends (Mott et al., 1985; Belasco et al., 1985). The data strongly implicate site-specific endonucleolytic cleavages in the 5’ region of the tat-ompA transcript. It was also observed that the upstream products of these cleavages were significantly less abundant than the downstream products.

The tat-ompA transcript was also analyzed using a seg- mented single-stranded Ml3 ompA probe (von Gabain et al., 1983) to monitor the decay of different mRNA segments. Apart from DNA fragments already described, no

additional major transcript endpoints were identified by

Regulation of E cd ompA mRNA Slablllty 897

872-

118-

72- l -

411)

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Figure 6 Sl Nuclease Analysis of fat-ompA mRNA with the “EcoRI” Probe

At 6 men after IPTG InductIon, RNA from E co11 BRE50 contalmng pTac- ompA was annealed with :he “EcoRI” probe (Figure 1). Hybrids were treated with Sl nuclease and analyzed by 6% denaturing pclyacryl- arnlde gel electrophoresls using a radlolabeled Haelll digest of 1~x174 as markers (sizes in nucleotldes at left) u. v. w, and y lndlcate the major Sl-reslstant DNA fragments. The sizes of the fragments in nucleotldes are as follows. y, 77 +- 2, w. 101 2 4; v, 115 ? 5. and u. 136 & 6 ’ corresponds to the full-length tat-ompA mRNA.

this method (data not shown). These results confirm that the smaller ompA-specific band seen in the Northern blot can be explained entirely by the 5’ endonucleolytic cleavages.

Cleavage of Wild Type ompA mRNA at Different Growth Rates The stability of ompA mRNA depends on the growth rate of cells. mRNA half-life is reduced when cell generation time is increased and consequently the steady-state con- centratron of ompA mRNA is reduced (Nilsson et al., 1984). In our hands, the wild type ompA mRNA has a half- life of 5-7 min for growth in MOPS-succinate (generation time, about 160 mm) and has a half-life of 20-25 min for growth in Luria broth (L broth) (generation time, about 40 min) (see Figure 8, Table 1). We took advantage of this growth-rate dependent stability of ompA mRNA to test the hypothesis that stability of the message follows the rate of 5’ endonucleolytic cleavage.

RNA samples isolated from E. coli C600 cells (express- ing the wild type ompA gene) cultivated at different growth rates were subjected to Sl nuclease analysis using the “Avall” probe (Figure 7). The Sl-resistant DNA fragments 6, C, D, and E identified for kc-ompA mRNA were also found for the wild type ompA mRNA, but at much lower concentrations. When the ratio of cleavage products to full-length transcript was examined it was found to be 3 times lower In cells grown with a doubling time of 40 min

-*-

-B- AC----

LB MOPS LB MOPS LB MOPS

Figure 7. Analysis of Wild Type ompA and tat-ompA mRNAs under Different Growth Condttlons

(A) RNA was prepared from E. co11 0300 cells growing in L broth (LB) or MOPS-succlnate (MOPS) and annealed with the “Avall” probe Hybrids were treated with Sl nuclease and analyzed by 6% denaturing polyacrylamide gel electrophoresls. Capital letters indicate cleavage products. and ’ Indicates the full-length ompA transcript. In this expertment the amount of total RNA used was that glvlng equal signal strength for the full-length transcripts from the two growth condltlons (B) Reverse transcnptase analysis of wild type ompA mRNA. RNA samples (the same as in Figure 7A ) were annealed with the tadlola- beled DNA ollgomer and transcribed with reverse transcnptase, and the DNA fragments were analyzed by 6% polyacrylamlde gel electrophoresls In this experiment the same amount of total RNA W&S loaded for the two growth condltlons. (C) Sl analysis of tat-ompA mRNA with the ‘Iwall” probe RNA samples were Isolated 6 men after IPTG Induction from E coli BRESO contalmng pTac-ompA Cells were grown I” L broth (LB) or in MOPS- succlnate (MOPS) The RNA samples were annealed with the ‘Avall” probe, treated with Sl nuclease, and analyzed by 6% denaturing polyacrylamlde gel electrophoresls Capital letters Indicate cleavage products. and * Indicates the full-length tat-ompA transcript

The DNAfragments B, C, D. and E were found to be Identical for both the wild type and tat-ompA mRNAs (by comlgration). DNA fragment A was detected only for the tat-ompA mRNA

(in L broth) than in those with a doubling time of 160 min (in MOPS-succinate) (Figures 7A and 76). This result confirmed the previous finding that the amount of full-length transcript per total RNA depends on its half-life (Nilsson et al., 1984). Interestingly, the amounts of cleavage products per total RNA were more or less the same under the two growth conditions, although the ratios of cleavage products to full-length transcripts differed by a factor of 3. In summary, these data show that the ratio of cleavage products to full-length transcript, as well as the amount of full-length mRNA, is affected by the growth rate. In contrast, the amount of the various cleavage products does not vary with growth rate.

Cleavage of tat-ompA mRNA at Different Growth Rates The effect of growth rate on the 5’ endonucleolytic cleavage of fat-ompA mRNA was studied by comparing the half-lives of tat-ompA mRNAs in cells grown in L broth versus MOPS-succrnate. We noted that the stability of the tat-ompA mRNA was reduced when cells were cultivated in MOPS-succinate medium (about 2.25 min in L-Broth

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100

10

100

10

1

0mpA

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I 1 I I I I

0 10 20 30 40 50 min

tat 0mpA

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Figure 8 Decay of the Wild Type and fat-ompA mRNA in E. coli at Different Growth Rates

(Top) Semllogarlthmlc plot of the decay of the 200 nucleotlde segment of the ompA wild type transcript. RNA was Isolated at the lndlcated time points after the addltlon of rifamplcln, and was hybridized wth the single-stranded DNA probe fragments as described prewously (van Gabain et al., 1983) The decay of the Sl-resistant, 200 nucleotlde segment (correspondmg to the 5’ end) was analyzed and plotted Closed circles reflect the Intact RNA remalnmg for cells grown in L broth (O/o), and asterisks reflect the Intact RNA remaining for cells grown II- MOPS-succlnate (“/a). (Bottom) Semllogarlthmic plot of the decay of the 295 nucleotlde 5’ DNA segment of the tat-ompA transcript. After IPTG InductIon and rlfamplcln addition, RNA was Isolated at the Indl- cated time points and analyzed by primer extension as described in Figure 4. The decay of the primer extension products was analyzed and plotted Closed circles reflect the Intact RNA remalmng for cells grown In L broth (o/o), and asterisks reflect the Intact RNA remalnlng for cells grown In MOPS-succlnate (o/o).

and 1.25 min in MOPS-succinate; Table 1, Figure 8). Thus, as with wild type ompA mRNA the decay of tac- ompA mRNA seemed to be growth-rate dependent, although with a half-life far shorter than that of the wild type message (Table 1, Figure 8). As we expected, Sl analysis

with the “Avall” probe showed that the ratio of cleavage products to full-length transcripts was also growth-rate dependent (Figure 7C).

The relative amounts of full-length transcript per total RNA, the ratios of cleavage products to full-length mRNA, and the half-lives are summarized in Table 1. The most striking conclusion is that the ratio of the cleavage products to full-length mRNA correlates well with the rate of degradation for both the wild type and fat-ompA mRNAs.

Discussion

In the present work we have shown that ompA mRNA in vivo is the target for growth-rate dependent endonucleolytic attacks. It has previously been shown that the half-life of ompA mRNA depends on the generation time of the cells, which could potentially explain the reduction in the rate of protein synthesis in cells with a longer generation time (Nilsson et al., 1984). The present work indicates that the half-life of this mRNA follows the rate of endonucleolytic cleavage. Such control of mRNA stability may provide a novel means of regulating gene expression.

The Various 5’ Ends of ompA mRNA Are the Result of Endonucleolytic Cleavage We found several different 5’ ends for tat-ompA mRNA. These cleavage products only appeared after induction of the tat promoter, strongly indicating that the cleavage products are the result of processing of the, full-length transcript. The endonucleolytic nature of the cleavages was confirmed by the identification of both upstream and downstream cleavage products. The 5’ end of each product was identified by three independent methods: Sl nuclease mapping, primer extension, and Northern blotting.

Most of the major cleavage points were located in the 5’ noncoding region of ompA mRNA. However, some cleavage points were also found In the coding region. Cleavage sites in the coding region of the mRNA are, we believe, less accessible, since they will be protected by ribosomes. They may also be less easy to detect, particu- larly if the resultant cleavage products have extremely short half-lives.

We also detected one cleavage site in the 54 additional 5’-terminal nucleotides of the tat-ompA hybrid transcript. Further experiments are needed to establish whether this cleavage site is susceptible to growth-rate dependent nuclease attacks in the same way as cleavage sites in the ompA transcript itself.

The Cleavages Seem to Control the Decay We suggest that the 5’ endonucleolytic cleavages provide the initial step in ompA mRNA degradation. This conclusion is based on the following results: First, the full-length transcripts are apparently processed to the cleavage products. Second, most of the detected cleavages reside in the 5’ noncoding region (a segment that has been found to be important for the stability of this mRNA species).

Regulation of E cd ompA mRNA Stablhty 899

Third, the ratio of cleavage products to full-length transcripts is proportional to the rate of degradation.

An alternative explanation for this last result is that the stability of the cleavage products is growth-rate dependent. We believe, however, that the cleavage products have the same stability at either growth rate. Equal amounts of cleavage products per total RNA were present at both growth rates. The amount of cleavage products is determined by the number of full-length transcripts processed to the cleavage products per unit time and the stability of the cleavage products. At equilibrium, the number of full-length transcripts synthesized per unit time, the number of full-length transcripts processed to cleavage products per unit time, and the number of cleavage products degraded per unit time are the same. We know that the synthesis of full-length ompA mRNA is not growth-rate regulated (Nilsson et al., 1984). Thus when we compare the two growth rates and find the same number of full- length transcripts synthesized per unit time as well as equal numbers of cleavage products, this must mean that the cleavage products are equally stable at both growth rates.

We observed the same correlation between the ratio of cleavage products and the rate of degradation for the tac- ompA mRNA. However, the number of cleavage products was higher after IPTG induction, indicating increased stability of the cleavage products in this situation. We explain this increased stability of the cleavage products by a retarded rate of translation (step time) of the fat-ompA mRNA, in response to overproduction. Such a slowdown of step time was previously found when there was overproduction of plasmid-encoded proteins (Pedersen, 1984). The rate of decay of the cleaved mRNA may well depend on the time it takes the last ribosome to exit the transcript. This interpretation is in agreement with former proposals that the decay of “Inactivated” mRNA keeps up with the last translating ribosome (Kennell, 1986). In a similar way we explain the short-term accumulation of the cleavage product E when there are high concentrations of rifampi- tin. While rifampictn did not seem to affect the stability of full-length mRNA, it is apparent that the decay of cleavage product E was retarded by rifampicin, an effect we believe due to a reduction in the rate of translation.

Both wild type and tat-ompA mRNAs exhibit growth- rate dependent stability. Such regulation could be attrib- uted to mechanisms that sense the altered concentrations of the OmpA protein. The present results argue against such a mechanism. IPTG induction increased the level of OmpA protein by about one order of magnitude, yet this increase was not followed by a change in tat-ompA mRNA stability.

The Importance of the 5’ Noncoding Region for mRNA Stability Endonucleolytic cleavages that control the degradation of mRNA have only been clearly verified in one case, the cleavage of the 3’-terminal hairpin structure of the phage lambda int message by RNAase Ill (Schmeissner et al., 1984). Recent reports have shown the importance of the

5’ noncoding region in controlllng mRNA stability (Gorski et al., 1985; Belasco et al., 1986). For example, when the S’noncoding region of the p-lactamase transcript (half-life, 2.5 min) IS replaced by the same region of the ompA message (half-life in rich medium, 15 min), the resultant hybrid mRNA is about 4 times more stable than the normal P-lactamase mRNA (Belasco et al., 1986). Moreover, decay of the (I-galactosidase mRNA has been suggested to be- gin at the 5’ end (Cannistraro and Kennell, 1985).

Wild type ompA mRNA decays at a uniform rate for all segments of the transcript when it IS derived from cells grown in MOPS-acetate (Nilsson et al., 1984). This sug- gests that, after the initial cleavages, decay ensues very rapidly. However, under conditions where the ompA mRNA has an extremely long half-life (e.g., when it is derived from cells grown in L broth), decay seems to tnitlate in the 3’ part of the message in the presence of rifampicin (von Gabain et al., 1983). On the other hand, in neither the former nor the present studies have cleavages in the 3’ part of the message been identified.

The fat-ompA Messages Are More Rapidly Degraded Interestingly, the tat-ompA messages are more susceptl- ble to degradation than the wild type ompA messages, pointing to further factors influencing the rate-limiting step of mRNA degradation. This cannot be the mRNA concentration itself, since the tat-ompA mRNA seems to have the same stability both before and after induction of cells with IPTG. One factor that might explain the accelerated degradation of fat-ompA mRNA is the cleavage within the 54 additional 5’-terminal nucleotldes of this transcript. At any rate, the 54 additional 5’.termtnal nucleotides are very likely to account for the shorter half-life o! the fat-ompA mRNA.

Models for Explaining the Role of the Cleavages There are several mechanisms by which 5’ endonucleolytic cleavages could initiate degradation of the ompA transcript. Most probably the cleavages disturb the loading of ribosomes onto the transcript, since some cleavages are in or next to the ribosome binding site. Previous reports have shown that depriving a transcript of its ribosomes destabilizes the transcript (Morse and Yanofsky, 1969; Schneider et al., 1978; Nllsson et al., 1987). More- over, such a hypothesis IS supported by previous findings that RNA sequences upstream of the ribosome binding site influence the loading of ribosomes (McCarthy et al., 1986).

After loss of nbosomes the transcript is open to second- ary endo- and exoribonucleases. An alternative model IS

that the cleavage sites could provide entry sites for a nuclease that would rapidly degrade the cleavage products. The two 3’-to-5’ exoribonucleases known in E. coli, RNAase II and polynucleotide phosphorylase, have been shown to be involved in mRNA degradation (Donovan and Kushner, 1986). However, none of the known enzymatic activities in E. coli can degrade RNA in the 5’.to-3’ dlrec- tion (Deutscher, 1985) Irrespective of the precise mecha- ntsm, the low concentrations of the cleavage products

Cell 900

indicate that they decay much more rapidly than the full- length mRNA.

Experimental Procedures

Bacterial Strains and Plasmids Host cells were E co11 K-12 strarns 0300 (Bachmann. 1972) and BRE50; the latter IS a derivatrve of MC4100 that falls to synthesrze ompA mRNA (Belasco et al., 1986).

The construct pTac-ompA was from plasmrds pTU100 (Bremer et al 1980) and pMMB66 (Furste et al , 1986). pMMB66 contarns the tat promoter, whrch can be repressed by the /acre gene product encoded by the same plasmid The Accl-Pstl fragment from pTU100. carryrng the ompA gene, was cloned between the Smal and Pstl sites of pMMB66, downstream of the tat promoter To make the ends compatrble, the Accl overhangrng end was frlled rn usrng the Klenow fragment of DNA polymerase I prior to digestion with Pstl and lrgatron After Inductron with IPTG. BFiE50 contarmng thus plasmrd grew normally untrl about 1 hr after addrtron of the Inducer (frnal concentrahon. 1 mM)

RNA Isolation Cells were grown rn L broth (generation hme, 40 mm) or MOPS-succrnate medrum (generatron trme, 160 mm) at 30°C as descrrbed prevrously (Nrlsson et al.. 1984). In mductron experrments, cultures were grown to an absorbance of 0.300 (at 650 nm) before the addrtron of IPTG. Transcription was InhibIted by the addrtron of rrfamprcrn (normally 200 nglml). and cell samples were collected at varrous time inter- vals and rapidly chrlled using crushed ice. Thereafter, RNA was prepared by the hot-phenol method, followed by a treatment wrth hrghly punfred DNAase I (von Gabain et al , 1983)

Northern Blotting, Sl Nuclease Mapping, Primer Extension, and Half-Life Determination Northern blot analysis was performed by fractronatron of RNA samples on a 0.8% agarose-formaldehyde gel followed by transfer to a nylon filter (Mamatrs et al., 1982). Hybndrzahon of filters with radroactrvely labeled DNA probes was performed as descrrbed prevrously (von Gabarn et al , 1983) Hybrrdrzatron of RNA and DNA at 45% and subse- quent Sl nuclease drgestrons were carrred out according to Berk and Sharp (1977).

For the reverse transcrrptase experrment we used a synthetic 24 nucleotide DNA olrgomer (5’.GACCAGCCCAGTTTAGCACCAGTG-3’) complementary to the ompA mRNA at the Avall site (Figure 1) The okgomer was 5’ end-labeled usrng polynucleotrde krnase and [y-a2P]ATF! A molar excess of this olrgomer was annealed to 5 ng of total RNA rn 50 nl of a 100 mM KCI solutron. Samples were heated at 90°C for 5 mm, slowly cooled to 42OC, adjusted to 10 mM Trrs-HCI (pH 83) and Incubated at 42% for a further 60 mm Subsequently, samples were adjusted to 5 mM of each dNTP 10 mM MgCla. 0.1% Nonrdet P-40 deter- gent, and 3 mM drthrothrertol (final volume 100 frl) and were rncubated with 3 U of reverse transcriptase at 42°C for 60 min.

Products of Sl nuclease dtgestron and primer extensron were analyzed by electrophoresis on 6% polyacrylamide-urea gels Autoradro- graphs were quantrtated by densrtometry as prevrously descrrbed (“on Gabarn et al., 1983).

For Northern analysis, m&translated pTU500 DNA, carryrng the ompA gene, was used as a probe (Freundl et al., 1985) The “Avall” probe (Figure 1) was obtained by digesting pTac-ompA DNA with Avall. labeling the 5’ends wrth [Y-~~P]ATP and T4 polynucleotrde krnase, and subsequently gel-punfyrng the labeled fragment correspondrng to the ompA 5’ region. The “EcoRI” probe (Frgure 1) was obtained by digesting pTac-ompA DNA at the unrque EcoRl site The linearized plasrmd was Incubated wrth T4 DNA polymerase for 1 mm at 37%. allowrng the overhangrng-end proxrmal regions to become srngle-stranded The smgle-stranded regions were reparred rn the presence of radroactrve nucleotrdes. usrng T4 DNA polymerase. The DNA was redrgested with BarnHI. whrch cuts at only a srngle site rn the plasmrd, and the fragment correspondrng to the ompA 5’ end was gel-purrfred.

Protein Analysis Fractronahon of proteins on 6% SDS-polyacrylamrde gels was carrred out as described by Mama& et al. (1982) The rdentrfrcatron of the mature OmpA protern on the gel was facrlrtated by antibody precrprtahon

Enzymes and Chemicals Restrrctron endonucleases were suppiled by Boehrrnger Mannhelm (Federal Republic of Germany). T4 DNA polymerase and T4 polynucleotrde krnase were supplied by New England BioLabs (USA). Sl nuclease. reverse transcnptase, nylon blotting filters, and radrorso- topes were obtained from Amersham (England) IPTG was purchased from Srgma Chemrcal Co. (USA). Rrfamprcrn was obtarned from Cuba- Gergy, Swrtzerland (Rrmactan). The DNA oligomer was synthesrzed by KabiGen, Sweden. Unless otherwrse stated, all reagents were used as recommended by the manufacturer

Acknowledgments

We thank Dr G. Nrlsson for theoretrcal and practical help and Tove Harde for expert technical assrstance We thank Drs. U. Henning and M. Bagdasanan for provrdrng plasmids, Drs. S. Molin. U. Lundberg, and S. Goldrn for crrtrcally reading the manuscrrpt. and Drs J Belasco and C. F. Hrggrns for strmulatrng discussions. Thus work was supported by grants from the Swedish Cancer Socrety (RMC), STU and SBT

The costs of publrcatron of thus artrcle were defrayed rn part by the payment of page charges This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to Indicate this fact.

Received January 12, 1987; revised January 4, 1988

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Site-specific endonucleolytic cleavages and the regulation of stability of E. coli ompA mRNA