Molecular Microbiology (1991) 5(8), 1961-1973

ADONIS D950382X9100214V

The rifampicin-inducible genes srn6 from F and pnd from R483 are regulated by antisense RNAs and mediate plasmid maintenance by kiiling of plasmid-free segregants A. K. Nielsen/ P. Thorsted,^ T. E. G. H. WagneH and K. Gerdes'* ' Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense f\A, Denmark. ^Department of f^icrobioiogy, Biomedical Centre. Uppsaia University, Box 581,S-751 23 Uppsaia. Sweden. Summary The gene systems srnB of plasmid F and pnd of plasmid R483 were discovered because of their induction by rifampicin. Induction caused membrane damage, RNase I influx, degradation of stable RNA and, consequently, cell killing. We show here that the srnB and pnd systems mediate efficient stabilization of a miniR1 test-plasmid. We also show that the killer genes srnB' and pndA are regulated by antisense RNAs, and that the srnC- and pntf&encoded antisense RNAs, denoted SrnC- and PndB-RNAs, are unstable molecules of approximately 60 nucleotides. The srnB and pndA mRNAs were found to be very stable. The differential decay rates of the inhibitory antisense RNAs and the killer-gene-encoding mRNAs explain the induction of these gene systems by rifampicin. Furthermore, the observed plasmid-stabiiization phenotype associated with the srnB and pnd systems is a consequence of this differential RNA decay: the newborn plasmid-free cells inherit the stable mRNAs, which, after decay of the unstable antisense RNAs, are translated Into killer proteins, thus leading to selective killing of the plasmid-free segregants. Thus our observations lead us to conclude that the F srnB and R483 pnd systems are phenotypically indistinguishable from the R1 hokJsok system, despite a 50% dissimilarity at the level of DNA sequence.

Introduction Post-transcriptional gene control may be exerted at Received 25 February, 1991: revised 17 May. 1991. 'For correspondence. Tel. (45) 66 15 86 00; Fax (45) 65 93 27 81.

several levels. One type of post-transcriptional control involves antisense RNAs. Antisense RNAs are small untranslated RNAs (50-100 nucleotides) that are complementary to the RNA molecule that is the target ot regulation, Usually (but not always), the antisense RNAs and target RNAs are encoded by the same DNA segment, and in opposite directions. Antisense RNAs are known to regulate a number of plasmid-, transposon- and phageencoded genes, and also several chromosomal Escherichia coii genes. For a recefit review of gene regulation by antisense RNAs, see Simons and Kleckner (1988). Post-transcriptional gene control may also be accomplished at the levei of messenger RNA stability (reviewed by Beiasco and Higgins, 1988). Most prokaryotic messenger RNAs decay rapidly, and the average mRNA half-life in E. coiiis of the order of one to two minutes (Pato etai. 1973). Some messages, however, such as the ompA and Ipp mRNAs, are much more stable, with half-lives of the order of 15-20 minutes (von Gabain ef ai, 1983; Peder-

sen etai, 1978). In 1972. it was observed that addition of rifampicin to certain E. coii strains led to degradation of stabie RNA (rRNA and tRNA) (Ohnishi and Schiessinger, 1972). Later it was shown that this degradation was due to the induction of an F-encoded gene near TniOOO that was designated srnB (Ohnishi, 1974; Ohnishi et ai, 1977). Degradation of stable RNA was also observed after addition of rifampicin to E. coli cells containing certain R factors (Ohnishi and Akimoto, 1980). The loci responsible for this phenotype were denoted pnd (promotion of nucleic acid sjegradation) (Akimoto and Ohnishi, 1982). Degradation of stable RNA after induction of srnB and pnd was dependent on the peripiasmic enzyme RNase I (Ito and Ohnishi, 1983). As the induction of the pndA and srnB systems was found to cause membrane damage and permeabilization, it was suggested that the stable RNA degradation was a secondary effect caused by RNasel influx through the damaged membrane. The R483 (Incl.,) and R16 (IncB) pnd and F srnB loci were cloned and sequenced (Ono etai, 1987; Sakikawa etai., 1989). Akimoto et ai. (1989) suggested that the srnB and pnd loci are regulated post-transcriptionally by an attenuator

1962

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R g . 1. Genetic organization ot the members ot the /rofc gene family, a, sm8(F);b.pnd(R483/R16):c, hofc/sofc (R1);d, fffn(F);e, ge/(£. co//);f. relF(E. coli) The seven saquenced memt>e(s of the rtofcgene family were aligned, using the start codons of the killer genes as the reference point. The names of the replicons encoding the killer loci are indicated m parentheses. The antisense RNA genes {srnC, pndB, sok, ffmB, and sof) are indicated wUh black bars bek)w the lines. The corresponding -10 and -35 sequences are also shown. The reading frames of the killer genes are shown as black bars above the lines. The overlapping srr^B. pndC. mok, flmC, and ort69 open reading frames are indicated with open bars. Positions (-10 and -35) of the assigned (srnB, hok, and flmA) and putative [pnd) transcription initiation signals of the killer mRNAs are indicated, and Ihe positions of Ihe corresponding 5' ends of the transcripts are indicated with arrows. Arrows in the 5' end ol the ge'and re/Floci indicate that transcription of the loci is initiated upstream of the region shown here. Transcription termination points are indicated by ttp. The arrow in the 3' end of the ge/locus indicates that transcription continues into an IS fd6 element, the beginning of which is shown with a cross-hatched bar(Pouls6n etai. 1989). The 3'end ofthe E. cotir^E gene is shown as a shaded box. The Figure was constructed by alignment of the nucleotide sequences of the loci (Gwttes et at., 1990b).

located upstream of the stnjctural killer genes. In this way, induction by rifampicin was explained as rifampicinpromoted readthrough of the attenuators. Recently, we showed that the srnB and pnd systems are structurally similar to the hokJsok killer gene system of plasmid RI (Gerdes ef ai, 1990b). A comparison of the plasmid-encoded and chromosome-encoded genes that constitute the hok gene family is shown in Fig. 1. The hoklsok system mediates plasmid stabilization by killing of plasmid-free segregants (Gerdes et ai, 1986a). The system encodes a very stable mRNA, the Hok mRNA. whose translation is inhibited by the unstable Sok antisense RNA. Sok antisense RNA is complementary to the translational initiation region (TIR) of the mok gene, the open reading frame that overlaps with the hok gene (Fig. 1). Detailed genetic analysis showed that Sok RNA regulates expression of hok by regulating transiation of the mok reading frame (T. Thisted and K. Gerdes, submitted). Thus the mok reading frame is an essential component of the hok/sok system, mo/c-homologous reading frames have been found in all of the antisense-RNA-regulated killer gene loci, including the smB and pnd systems (Fig.1). Translation of the stable Hok mRNA leads to production of the Hok protein, a potent cell-killing agent that kills bacterial cells from within by damaging the cell membrane (Gerdes et ai., 1986b). The membrane damage causes gross cellular changes, leading to cells with the characteristic 'ghost-cell' appearance (i.e. condensed cell poles and central clearing). Because of the structural similarities between hok/sok, srnB and pnd, we decided to clone and analyse the two latter systems. We show here that srnS and pnd mediate efficient plasmid stabilization. Furthermore, both systems encode very stable killer mRNAs and unstable, regulatory antisense RNAs. We suggest, therefore, that the smB and pnd systems mediate plasmid stabilization by killing of plasmid-free segregants, as has been shown for the R1 hok/sok locus. Results Cloning of the pnd locus from plasmid R483 and the srnB locus from piasmid F According to Ono ef al. (1987), the pnd locus of plasmid R483 is located on a 0.85 kb EcoRt-Sa/l restriction fragment. We cloned this DNA segment between the FcoRISal\ sites of pBR322. The ligated DNA was transformed to strain CSH50 and the cells screened for changes in morphology after addition of rifampicin. The cells of two clones displayed the typical "ghost-ceir morphology after rifampicin treatment. Both contained identicai plasmids carrying the R483-derived EcoRI-Sa/l fragment. The

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arnC R g . 2. Physical and genetic maps ot the pnd and smSloci from plasmids R483 and F, The Figure was based on the puWi^&d nucleotkJe sequence ot R483 pnd (Ono efa/., 1987) and F smfi(AkJmoto efa/., 1986) loci. The original co-ordinates of the nucleotide sequences have been preserved.

plasmid was designated pAN1, The authenticity of the pnd-encoding fragment in pANI was verified by DNA sequence analysis. The fto/t-homologous killer gene encoded by the pnd locus was previously designated pndA{¥\Q. 1 and Gerdes ef a/.. 1990b). A detailed physical and genetic map of the R483 pnd locus is shown in Fig. 2. The F srnB locus was cloned by a similar procedure, although in this case we used chromosomal DNA from an Hfr strain as starting material. The F smB locus is located on a 1.1 kb EcoRI-eamHI fragment (Onishi etai, 1981). Fragments of this approximate size were isolated after digestion of chromosomal DNA from strain Hfr H with EcoRI and SamHI. ligated to pBR322 (cieaved with the same enzymes), and then transformed into CSH50. The transformants were screened for the typical ghost-cell morphology after rifampicin treatment. Two positive clones were analysed further and both were found to contain pBR322 with the 1.1 kb EcoRI-SamHI restriction fragment. This plasmid was designated pPT490. The correct inserts were verified by restriction anaiysis and comparison with the previously published restriction map of the smS-encoding DNA fragment. The hofc-homologous

killer gene responsible for ghost-celi induction was designated srnB' (Fig. 1 and Gerdes etai, 1990b). A physical and genetic map of the srnB locus is shown in Fig. 2 (lower). The above results show that the pnd and smB loci are induced by rifampicin and that this induction results in gross cellular changes similar to the changes in morphology observed after RI ho/c expression. scnB anrfpnd stabilize mini-RI replicons ' The DNA fragments described above were cloned into an unstable mini-RI replicon which also carried the lac genes (pOU82). The presence of the lac genes on the test-plasmid provided an easy and time-saving plate assay for plasmid-containing cells. Thus, pAN3 is pOU82 (bia*) carrying the pnd locus on a 0.85 kb EcoRI-SamHI fragment, and pAN4 is the kanamycin-resistant [aphA*] derivative of pAN3. Similarly, pPT390 is the pOU82 vector-plasmid carrying the srnB locus on a 1.1 kb EcoRISamHI fragment, and pPT391 is the corresponding kanamycin-resistant derivative of pPT390. Plasmids used and constructed are listed in Table 4. Table 1 shows the loss frequencies of pOU82 and its

1964

A. K. Nielsen el a\.

Table 1. StabiFization ol pOU82 (mini-Ri) wttti smB, pnd and hokisak.

Plasmid designation

Killer locus present

Loss frequency per cell per generation

pOU82 pPT390 pAN3 pTT820

None smB from F pod from R483 hofc/sofc from R1

1 X 10"=

1

5x10^ 5x10"* 3x10^

200 200 300

Factor of stabilization

LF ^ t u e s were obtained as clescnbed previousty (Gardes el ai.. 1985). The cells were grown exponentially for at least 60 generations. Frequency ol plasmid-tcee cells were determiried by platirtg on (McConkey-lactose) indicator plates.

hok/sok-, pnd-, and smS-carrying derivatives. As seen from tfie Table, the R1 hok/sok system stabilized the miniRI repiiron about 300-fold, in agreement with previousty published data (Gerdes et ai, 1985). Interestingly, the pnd and smB loci both had quantitative and qualitative effects comparable to hok/sok with regard to the stability of maintenance of a mini-RI plasmid (Table 1). srnB and pnd express incompatibility The R1 hok/sok locus exerts stabilization-associated incompatibility (Rasmussen et ai, 1987: Table 2). The incompatibility is a consequence of the Sok-RNA-mediated repression of hok expression described below. The stabilization mechanism is dependent on rapid decay of the Sok-RNA in newborn ptasmid-free cells. Thus, the presence of a second, so/(-carryJng, plasmid in cells which have lost the stabilized test-plasmid inhibits expression of the killer protein and therefore prevents killing of the plasmid-free segregants. This ^rans-inhibition

Table 2. Incompatibility exerted by srnB, pnd and hok/sok.

Test plasmid (mim-Ri Kan")

Inc plasmid (pBR322)

pMH82 pPT390 isms') pPT390 (smB-) p A M (pndl pANA ipndl pAN4(pntf-) pTT720 {hok/sok') pTT720 {hok/sok')

pBR322 pBR322 pPT490 (smfl-) pBR322 pANI(pnd') pAN5 (pndS') pBR322 pPR633 {hok/sok-)

Loss frequer>cv of mini-RI

plasmid 1x10"^ 5xl(r* 1x10^ 5x10"* lx10r= 1x10-^ 5x10-* 1 xicr*

of kitler-gene expression leads to destabilization of the test-plasmid, and is usually detected as incompatibility on McConkey-lactose indicator plates. Because of the above, we tested whether the smS and pnd loci also displayed the incompatibility phenotype- The pAN1 plasmid (pBR322-pnd*^) was transformed into CSH50 carrying pAN4 (mini-RI-pnd*), and the stability of pAN4 was measured. As seen from Table 2. the presence of pnd on a second plasmid destabilized pAN4. The vector-plasmid pBR322 had no such effect (control). The gene encoding the incompatibility determinant was designated pndB. Likewise, the srnB locus also expressed incompatibility (Table 2). The gene responsibie for the incompatibility phenotype was designated srnC. Taking the structural similarities into consideration, these results strongly suggest that the hok/sok. pnd and srnB loci stabilize the mini-Ri replicon by the same moiecuiar mechanism.

Cloning of the gene (srnC) which specifies the smBassociated incompafibility determinant The DNA fragment extending from the Kpnl site (+i96) to the Sspl site (+398) in the srnB locus (Fig. 2) was cloned between the Kpni and H/ndll sites in the pGEM3 and pGEM4 high copy-numt>er vectors, thus resulting in pPT3 and pPT4, respectively. Next. pPT3 and pPT4 were transformed into CSH50 carrying pPT391 (mini-RI-smS*). The presence of pPT3 or pPT4 resulted in a complete destabilization of pPT391, Thus the srnC gene is located within the region from +196 to +398 in the srnB locus. This region encodes part of the srnB mRNA leader and the 5' part of the smB structural gene, including the smB translational initiation region. In analogy with the RI hok/sok system, we therefore expected the srnC gene product to be an antisense RNA complementary to the smS mRNA. The srnC gene product is a small, metabolically unstable antisense RNA

Factor of stabilization 1 300 1 300 1 1 200 1

Heteroplasmid populations were generated by transformation of the pBR322 derivatives into CSH50 containing the mlni-Rl test-plasmid in question. Selection was on dOLtble selective p>lates. Next, the LF values (toss frequerKies) o( ihe mini-Ri test piasmids were obtained as in a usual plasmid-stability test (Gerdes et al.. 1985) Selection was maintained for the incomir>g pBR322 derrvattves dunng the measurement

Plasmid pPT4 was transformed into the rifampicin-permeable strain AS19. and RNA was prepared from this strain before and after the addition of rifampicin. Figure 3 shows a Northern-transfer analysis of this RNA. The srnC probe used was single-stranded RNA extending from +196 to 398, with the same polarity as the smSmRNA. As seen in Fig. 3. pPT4 produced an antisense RNA of -60 nucleotides (nt). We designated this RNA species SrnCRNA. The SrnC-RNA decayed rapidly after rifampicin. with a half-life of one to two minutes. Curiously, a stable RNA of -120 nt was also detected with the SrnC-probe in AS19 (ASI9 is an E. coli B denvative) but not in E. coli K12.

Antisense RNA-regulated killer genes srnB and pnd

1 1

3

5

10 MW nt 205

117

27

Fig. 3. Identification and stability of the SrnC antisense RNA visualized by Northern analysis,Total RNA was prepared from strain AS19/pPT4 (pGEM4-s/-nH') before and after addition of rifampicin and processed as described in the Experimental procedures. Time points o( sampling (minutes) are indicated above each lane, and 20 MS o' I^NA was a;^lied per lane. The probe used was generated by in vitrc transcriplion using T7 RNA polymerase arnJ pPT3 plasmid DNA as template. The stable RNA appearing as a ~ 120 nt band was also detected in a plasmid- free background, and therefore represents hybridtzalion to cellular RNA.

pndB probe used was a single-stranded RNA extending from +225 to +329, and with the same polarity as the pndA mRNA. As seen, the pANI- and pAN5-carrying strains produced an antisense RNA of an apparent size of -60 nt. We call this RNA species PndB-RNA. In pAN5, the expression of the antisense RNA was three- to fourfold higher than in the case of pANI in accordance with the higher copy number of the latter plasmid (pBR322 versus pUCi9). The PndB-RNA decayed rapidly after addition of rifampicin (with a half-life of approximately one minute; Fig. 4). In Fig. 4 (lane 9) we included unlabeiled, purified in vifro Sok-RNA of 67 nucleotides (see below). As seen, the PndB probe used here resulted in a weak hybridization signal with the Sok-RNA. On the level of RNA sequences, there is approximately 60% identity between the PndB probe and the Sok-RNA, which perhaps explains this unexpected hybridization signai. Incompatibility expressed by hok/sok, pnd and smB is allele-specific The cross-hybridization between the PndB probe and the Sok-RNA described above prompted us to ask whether the antisense RNAs also interacted at the functional level in vivo. To test this, we investigated whether any of the hok/sok, pnd or srnB systems could destabilize any of the heterotogous systems. As can be seen in Table 3, the

2

Cloning of the gene fpndBJ which specifies the pndassociated incompatibility determinant The DNA fragment extending from the Acd site (+225) to the Sfaf^\ site (+329) in the pnd tocus (Fig. 2) was cloned into the Smal site of pUCi9, resulting in pAN5. Next, pAN5 was transformed into CSH50 carrying pAN4 (miniRI-pnd*). The presence of pAN5 resulted in a complete destabilization of the pAN4 piasmid. Other DNA fragments derived from the pnd region had no such effect (not shown). Thus, the pndB gene is located within the region from +225 to +329 in the pnd locus. This region encodes part of the pndA mRNA leader and the first part of the pndC structural gene (Fig. 2), Because of the above, we expected the pndSgene product to be an antisense RNA complementary to the pndA mRNA.

The gene product of pndB is a small, mefabolically unstable antisense RNA Pfasmids pAN1 and pAN5 were transformed into strain ASI 9, and RNA was prepared from these strains. Figure 4 shows a Northern-transfer analysis of this RNA. The

1965

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0

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153

PndB

24 Fig. 4. Identification and stability of the R483 PndB anlisense RNA using by Northern analysis,Total RNA was prepared from strains ASi9/pAN1 (pBR322-pnd') and ASi9/pAN5 (pUC19-pndS') before and aflet addition of rifampicin and processed as described in the Experimental procedures (lanes 2--6), Time points of sampling are indicaled (minules). Usually. 2 0 M 9 of RNAwas applied to each lane. Lanes 1 and 8 are molecular weight mafkers. Purified /n-vi/ro-synthesized Sok-RNA was included in the analysis (lane 9), The stabie RNA appearing as a -70 nt band was also detected in a plasmid-free background, and therefore represents hybridization to cellular RNA.

1966 A. K.Nielsen etal Table 3. Test for cross incompatibility between hok/sck. pnd and smB. Test-plasmid (mini-Ri Kan")

Inc plasmid (PBR322)

pPT391 {smB') pPT391 {srnB') pPT902 {smB') pAN4 {pnd') pAN4 {pfKr) pAN4 {pnd^) pTT720 ihok/sok-) pTT720 (hok/stOi-) PTT720 (hok/sok')

pPT490 {smB') pPR633 (hoWsofc*)

Incompatibility' (destabili2ation of lest-pl.)

pANI {pnd') pANl (pnd-) pPR633 (hoWsok*) pPT490 {smB') pPR633 (ftoWsc*-)

pANi (pnd') pPT490 (srnS-)

4 + + -

a. Incompatibility was measured by transfonnatjon of the pBR322 derivatives (Ap") into CSH50 carrying the mini-RI Kan" lest-plaamid In question and subsequent streaking on non-selective (McConkey-lactose) indicator plates. A plus sign indicates the detection of plasmid-tree (Lac~) colonies in the test.

presence of the hok/sok system on pBR322 (pPR633) had no destabilizing effect on the mini-RI -pnd* (pAN4) or the mini-RI-smS* (pPT902) plasmids. In fact, neither of the systems seemed to be able to destabilize any of the others. Thus the antisense RNAs did not seem to crossreact, and we conclude that the incompatibility expressed by the hok/sok, pnd and srnB systems is ailele-specific. This result indicates that the antisense RNAs cannot interact with the heterologous mRNAs.

The srnB and pnd mRNAs are very stable As described previousiy, the high degree of stability of the hok mRNA is a crucial element in the mechanism leading to activation of hok mRNA translation after decay of the inhibitory Sok-RNA. Figures 5A, B and C visualize the anB, pndA and hok mRNAs before and after rifampicin. The hok mRNA blot was included for comparison. As can be seen, all three mRNAs appeared equally stable and had very long half-lives. Interestingly, new and shorter mRNA species appeared after rifampicin addition in all three cases (see the Discussion). Approximately 30 minutes after addition of rifampicin, the smB- and pnd-containing cells changed into the characteristic ghost cells, as was previously described for the Ri hok/sok system (Gerdes era/., 1990a). Comparison of the antisense RNAs encoded by the plasmid-borne killer gene loci The above results showed that the SrnC and PndB RNAs are characterized by similar sizes and stabilities relative to Ri Sok RNA. To determine the exact sizes of this family of antisense RNAs, the RNAs were synthesized in vifro using E. coli RNA potymerase and supercoiled plasmid DNA as templates. The result of such an experiment is

shown in Fig. 6. From a DNA-sequencing ladder present on the same gel we were able to determine the exact sizes of the in vitro products. Thus the SrnC RNA was 63 nt long, the PndB RNA was 66 and 65 nt long, the FlmB RNA was 61 and 60 nt long, and the Sok RNA was 66 and 67 nt iong. The flmS-carrying clone, pNLW2, is a pUC19 derivative with the ho/(-homologous flm locus of the F plasmid on a 880 bp EcoRI-SamHt fragment (S. Motin, unpublished). In conclusion, all the plasmid-encoded antisense RNAs have similar sizes of 60-67 nt.

Discussion In this work we show that the srnB system of F and the pnd system of R483 mediated efficient stabilization of a mini-Ri test plasmid and that the systems expressed allele-specific incompatibility. The incompatibility phenotype suggested that the srnB and pnrf systems produced (rans-acting factors that prevented the expression of the stabilization phenotype. In line with this, we also showed that the srnB and pnd loci code for unstable antisense RNAs of -60 nt that seem to be the mediators of the observed incompatibility. The srnC and pndfl genes, which encode the SrnC and PndB RNAs, were mapped to the region encoding the leader of the SrnB and PndA mRNAs (Fig. 1). This suggests, as has been shown in the case of Ri-Sok RNA, that the SrnC and PndB antisense RNAs inhibit translation of their cognate killer mRNAs. In line with this, the incompatibility expressed by the antisense RNA genes must be a consequence of this inhibition (see below). The SrnB and PndA mRNAs were found to be extremely stable, with half-lives in the order of hours (Fig, 4A and 4B), Thus the srnS and pnd systems encode very stable killer mRNAs and unstable antisense RNAs that are complementary to the killer mRNAs. These results strongly indicate that the srnB and pnd systems mediate piasmid stabilization by the same mechanism as that shown for the hok/sok system: cells, which at cell division lose an srnB- or pnd-carrying plasmid. experience a rapid decay of the corresponding antisense RNA, thus leading to translation of the stable kiiler mRNA still present in the plasmid-free segregants. The killer protein (SrnB' or PndA) is then produced, and the plasmid-free cells are killed. In view of this simpie model the incompatibiiity exerted by the antisense RNA genes (i.e. srnC and pndB) present on a co-existing plasmid is readily explained: the continuous synthesis of the antisense RNA in cells which lose the killer gene-carrying plasmid prevents the activation of translation of the stable killer mRNA in these cells. This continuous inhibition leads to repression of the killer system, which is phenotypically detected as destabilization of the plasmid that carries the killer system. As in the case of the hok/sok system, we suggest that the

Antisense RNA-regulated killer genes srnB andpnd

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-27 Ftg. 6. In vitro synthesis of the SrnC, PndB, FlmB aixl Sok antisense RNAs, The antisense RNAs were synthesized in an in v/fro standard reaction using E. coli RNA polymerase and supercoiied plasmid DNA as templates. The templates were: SrnC-RNA (pPT4); PndB-RNA (pANI): FlmB-RNA (pNLW2); Sok-RNA (pPR633), The molecular weight standard is irxlicated in the Figure. A sequencing ladder was included in the same gel (not shown).

antisense RNAs inhibit translation of the reading frames (srnB and pndC) that overlap with the srnB' and pndA killer genes (Fig. 1). Like the situation with pndA and srnB' genes, R1 hok expression was induced by rifampicin (Gerdes ef al., 1990a). In the case of the hok/sok system, a new and shorter Hok mRNA species appeared after addition of rifampicin (Fig, 4C). Appearance of the new Hok mRNA species was found to be correlated with expression cf the hok gene (Gerdes efai, 1990a). We therefore postulated that it was the shorter Hok mRNA species that was transiationally active, both after the addition of rifampicin, and in newborn plasmid-free cells. As is seen in Figs 5A and B, new and shorter SrnB and PndA mRNA species also

appeared after the addition of rifampicin. These clear observations indicate that although the hok/sok, smB anti pnd sequences are quite different (-50% dissimilarity; Gerdes et ai, 1990b), the three killer gene systems are regulated by very simitar mechanisms. We are currently investigating this regulation further. Ohnishi s grcup discovered the pnd and smB loci because of their induction by rifampicin (Ohnishi and Schiessinger, 1972; Ohnishi ef ai. 1977; Ohnishi and Akimoto, 1980). Recently, these authors suggested that the smB and pnd genes are regulated by transcriptional attenuators located just upstream of the killer gene reading frames (srnB' or pndA). In keeping with their model, the rifampicin-induced expression of the Killer genes was explained by rifampicin-promoted readthrough cf the attenuators, thus leading to a complete and translatable transcript after addition of rifampicin (Akimoto et al., 1989). The attenuator model is not supported by the fact that full-length SrnB and PndA transcripts are present before the addition of rifampicin (Figs 5A and 5B), Therefore we suggest that the induction of the srnB and pnd systems by rifampicin can be accounted for solely by a rapid decay of the SrnC and PndB antisense RNAs and subsequent translation of the stable SrnB and PndA mRNAs (truncated or full-length mRNAs). The antisense RNAs encoded by the smB, pnd, flm, and hok/sok systems have similar sizes (Fig. 6). The 5' ends of the antisense RNAs are known in the cases of Sok (Gerdes et ai, 1988), FlmB (Loh ef al., 1988). and Sof (Poulsen et aL, 1991). The 5' ends of the SrnC and PndB RNAs were deduced from putative promoter structures present in the DNA (Gerdes etal.. 1990b), The 3' end of the Sof RNA was deduced from the transcriptional terminator structure present in the DNA. On the basis of these data and the nuclectide sequences, we suggest that the primary structures of the antisense RNAs are as shown in Fig. 7. The antisense RNAs shown in Fig. 7 could be folded into strikingly similar secondary structures that conformed to the following general stnjcture in ali cases; a 5' end non-structured leader of 8-16 nudeotides, a GC-rich stem of 17-23 base pairs, and an antisense RNA 'recognition-loop' of six to eight nucleotides. Furthermore, all the antisense RNAs have one or more mismatches in the stem located four to six nucleotides from the beginning of the loop. Unpaired bases important for antisense RNA:target RNA interaction have also been found a few nucleotides from the recognition loops for the CopA RNA of RI and RNA I of ColEI (Tomizawa etai, 1981; Wagner and Nordstrom, 1986; Persson etai, 1990), According to the general scheme for antisense RNA.target RNA interaction, the RNA molecules form an initial reversible contact between the complementary recognition loops ('kissing'), followed by nucleation of the

Antisense RNA-regulated kilter genes srnB and pnd

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1969

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°U-A G-C C-G U-G. C-G C-G G-C G-U G-U G-U A-U C-G. 5•-GUUGUCUAAGCAUG-UCC-3

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Sok-RNA from plasmid RI (67 nucleotidea|

FlmB-RNA from plasmid F (61 nucleotides)

SrnC-RNA from plasmid F (63 nucleotides)

A-U A-U U*A U-A

G U

A-U A-U U-A

G-Cn .G-U

.U-A

U-A

G-C G-U

G-C.

G-0 U-A G-C C-G U-G C-G C-G • G-CM

U-A.

A-U

A A.

A A U A G

A A A

A A U A A A G A.

U-A U-A A-U

U-A U-A

e-c u u u

A A .G-U

U-A A-U

-A-U iiG-C

G-C.

U-G G-C G-C G-U U-G

.G-C. G-C U-A U-G C-G C-G G-C A-U G-U .A-U.

A A U G

C-G.

U-A G-C G-U G-C C-G

C-G

.U-G

.C-G

C-G C-G G-C

G-C G-U G-U

G-U.

A-U

5•-GUUGUCUAGGCAUUACA-UGCUG-3

U 1 . G-C 5•-GUUGAGGCAUUACA-UGCUACAUG-3

. G-U 5•-GUUCAGCAUAUAG-U-3

PndB-RNA from plasmid R483 [66 nucleotides)

PndB-RNA from plasmid R16 [66 nucleotides)

Sof-RNA from E. coli (53 nucleotides)

1

.

"G-C

U

1

Fig. 7. Primary and proposed secondary stnjctures of the antisense HNAs encoded by the members of the hok gene family. The primary sequences were from Gerdes etal. (1990a) (Sok); Loh etaL (1988) (FlmB): Ono etaL (1987) (PndB from R483): Sakikawa elal. (1989) (PndB from R16); Akimoto elaL (1986) (SmB); and Poulsen etal. (1989) (Sof). The secondary structures of the RNAs shown were calculated using the computer programme FOLD developed by Zuker and Stiegler (1981).

5' ends of the antisense RNAs to the target RNAs (Tomizawa, 1984; Persson et ai, 1988. Kittle et ai, 1989). Nucleation eventually proceeds tc the formation of a complete duplex (Tomizawa. 1984; Persson ef al., 1988). We suggest that the initial interaction between the antisense RNAs shown in Fig. 7 and their cognate target mRNAs occurs between the antisense RNA recognition loops and corresponding loops in the target mRNAs. Obviously the killer mRNAs (Hok. FlmA, PndA. SrnB)

are activated after decay of the respective antisense RNAs (Gerdes ef ai, 1990a; this work). We are presently investigating the mechanism by which these mRNAs circumvent the problem of irreversible inactivation by the antisense RNAs. The new mRNA species that appear after rifampicin addition may at least partly answer this question. As the formation of the truncated mRNAs is inhibited by streptolydigin. we cannot at the moment exclude the possibility that these mRNAs are formed by

1970

A. K. Nielsen et al,

Table 4. Bacterial strains, plasmkls used and constructed.

Strain/ Plasmid

Relevant genotypes'

AS19 CSH50 MC1000 C600/R483 HfrH PUC19 PBR322 pGEM3 pGEh14 PGEM154 PGEM342 pPH633 pOU82 pMH82 pAN1 |WM2 pANS pAN4 PANS pANB pAN7 pPT3 FiPT4 pPT490 pPT390 pPT391 pTT720 pTT820

Rit permeabte .Mlao-pmt Mlacf) Tnmetfioprim" Hfr strain Ap" Ap"Tc" Ap"pT7 Ap"pT7 Ap"pT7 Ap"pT7 Ap" Ap" Lac" Kan" Lac' Ap" Ap" Ap" Kan" Ap" Ap"pT7 Ap"pT7 Ap"pT7 Ap"pT7 Ap" Ap" Kan" Kan" LacAp" Lac-

Killer system present (coordinates)"

pnd

smB

hok/sok (-300 to +330) ftofVso'i(+342to+580) hok/sok {+^ to+580)

pnd (-100 to+756) pnd(-100to+756) pnd(-100to+756) pnd (-100 to+756) pnd(+225to+329) pnd(+225to+329) pnd(+330 to +756) smfl(+196to+398) smfl (+196 to+398) smfl(+1 to+1100) smfl(+1 to+1100) smfl(+1 to+1100) hoklsok{*^ 10+580) hoklsok (+^ 10 +580)

Reference/Source Sekiguchi and lida (1967) Miller (1972) Casadaban and Cohen (1980) Strain collection Strain collection Yanlsch Penx>n etal. (1985) Bolivar efaA (1977) Promega Biotech Promega Biotech Gerdes era', (1990a) Gerdes era/, (1990a) Rasmussen elal. (1987) Gerdes fff a/. (1985) This work -

a. Ap*^: ampicillin resislance, Tc": letracycline resistance; Kan": kanamydn resisianca: pT7: bactenophage T7 RNA polymerase responsive promoter. b. Co-ordinates of ttie ktlker systems accordir^ to the published sequences: hokJsok. Gerdes st al. (1990a): smB, Akimoto etal. (1986);pnd, Ono etal. (1987).

de novo RNA synthesis. The recent discovery of putative RNA helicases in E. co//(Iggo etai, 1990) has presented the possibility that the killer mRNAs couid be activated by an 'unzippering' mechanism. In this paper we show that all the plasmid-encoded hokhomologous kiiier genes seem to be regulated by small, metabolicaliy unstable antisense RNAs. The chromosomal gef gene may be regulated by a similar control loop. The discovery that these prokaryotic killer genes are regulated by similar control loops should tacilitate a more detailed analysis of the underlying molecular mechanisms.

gene in a translational fusion with the deoC gene (without the deo promoters) and the lacYA genes (Gerdes et ai, 1985), Thus pOU82-containing cells give rise to colonies that are weakly Lac* on McConkey-lactose plates and clearly blue on the more-sensitive X-Gal (5-bromo-4-chloro-indolyl-[J-D-gatactoside) plates. In addition. pOU82 contains unique, juxtaposed EcoRI and BamHI restriction sites useful for insertion of DNA fragments.

Construction of plasmids

BactdflBl strains and plasmids

Plasmid pMH82. A 1.2 kb Pst\ fragment encoding Ihe kanamycin-resistant aphA (Kan") gene from Tn903 was inserted into the Psfl site in the bla gene of pOU82, resulting in pMH82, As the pOU82 vector contains five Pst\ sites, the vector was cut partially with this enzyme, and the largest linearized fragment was recovered prior to ligation. Potentially positive clones were selected by means of the Kan". Ap^ (ampicillinsensitive) phenotype.

The E. coli K-12 strains used are listed in Table 4 together with the plasmids used and constructed. The plasmids used in addition to the standard cloning vectors pBR322 and pUCi9 are described below, Plasmid pPR633 is a p8R322 derivative carrying the hok/sok locus on a 580 bp EcoRI-SamHI fragment (Rasmussen et ai, 1987). Plasmid pOU82 was derived from the mini-R1 'runaway replication' vectors originally developed by Larsen etai (1984). The pOU82 plasmid contains the IacZ

Plasmid pANI. DNA of the resistance factor R483 (90 kb) was digested with EcoRI and Sa/I and fragments in the range 0.5-1.2 were purified from an agarose gel using DEAE filter paper. These DNA fragments were ligated into pBR322 restricted with the same enzymes. Transformation and subsequent screening for ghost celis after rifampicin yielded pAN1. The DNA inserted into pANl was a 0.85 kb EcoRI-Sa/l fragment encoding the R483 pnd locus.

Experimental procedures

Antisense RNA -regulated killer genes srnB and pnd Plasmid pAN2. The 0.85 kb EcoR\-Sal\ fragment containing the pnd locus was blunt-ended using Klenow potymerase (large fragment) and inserted into the Smal site in the potyiinker region of pUCI 9. Thus the 0.85 kb pnd^ fragment of pAN2 was then flanked by EcoRI and SamHI restriction sites. Plasmids pAN3 and pAN4. The 0.85 kb EcoH\-BamH\ pnd' fragment of pAN2 was inserted into pOU82 (Ap") and pMH82 (Kan"), thus resulting in pAN3 and pAN4, respectively. Plasmid pAN5. The 105 bp Acc\~SfaH\ (+225 to +329) pnd fragment was purified from an agarose gel, and blunt-end-ligated into the Smal site of pUC19. Plasmid pPT390. The EcoRl-SamHI fragment of pPT490 was inserted into pOU82. Thus. pPT390 is a bla* lac* srnB* mini-R1 replicon. Plasmid pPT391. The EcoRl-SamHI fragment cf pPT490 was inserted into pMH82. Thus, pPT391 is isogenic with pPT390 except for the aphA gene (Kan") inserted into the bla gene. Plasmid pPT490. Chrcmoscmai DNA from Hfr H was digested with SamHI and FcoRI. and the fragments separated on an agarose gel. Fragments in the range frcm 0.7-1.3 kb were purified and ligated to pBR322 also restricted with EcoRI and SamHI. The ligation was used to transform CSH50, and the transformants were subsequently screened for ghost-cell formation after addition of rifampicin (see the Results). Positive clones contained pBR322 with the 1.1 kb EcoRI-SamHI srnB fragment. Plasmid pTT720. The 580 bp SamHI-EccRI fragment from pPR633 carrying the hok/sok system was inserted into the EcoRI-SamHI sites of pf^H82, yielding pTT720. Plasmid pTT820. The 580 bp SamHI-EcoRI fragment from pPR633 was inserted into the EcoRI-SamHI sites cf pOU82, resulting in pTT820.

Plasmids used for the in vitro generation of singlestranded RNA probes The cIcning vectors pGEM3 and pGEM4 contain opposing 17 and SP6 RNA polymerase promoters adjacent to multiple cloning sites (mcs), such that DNA fragments inserted into the mcs can be transcribed by the RNA poiymerases. pGEM3 and pGEfVI4 were used in the following constructions as described. PlasmidpAN6. The 123 bp EcoRI-SamHI fragment carrying the pndB* gene from pAN5 was inserted into pGEM3, thus resulting in pAN6. Transcription from the T7 RNA polymerase promoter of pAN6 produces a single-stranded transcript complementary to the pndS antisense RNA. Plasmid pAN7. A 427 bp S/aNt-SamHl fragment (+330 tc +756) carrying the pndA gene was inserted into pGEM4. Transcription from the T7 promoter of pAN7 yields a singlestranded RNA complementary to the pndA mRNA.

1971

Plasmids pPT3 and pPT4. The 202 bp KpnI-Sspl (+196 to +398) srnB fragment was purified from an agarcse gel and ligated to pGEM3 and pGEM4 restricted with Kpnl and H/ncll, resulting in pPT3 and pPT4, respectively. These plasmids were used for the generation cf single-stranded RNA probes by in wYro transcription using T7 RNA pclymerase, pPT3 yielded an RNA probe that was complementary to SrnC RNA, and pPT4 yielded an RNA probe that was complementary to SrnB mRNA.

Biochemical methods Preparation cf plasmid DNA, and DNA manipulations were according to Maniatis etai (1982). in vitro RNA synthesis was performed according to the manufacturer (Promega Biotech). In vitro transcripticn reactions and purification of RNA were performed essentially as described by Persson et ai (1990). Generation of single-stranded RNA probes for Northern analysis was accomplished using T7 RNA polymerase and the pGEM vectors from Promega. In vitro transcription with E. ooli RNA polymerase was acccmplished essentially as described by Persson e/a/. (1988).

Genetic methods Stability and incompatibility tests were accomplished as described previously (Gerdes et ai, 1985). Antibiotics were added at the following standard concentrations: ampicillin, 100 |ig ml"'; kanamycin, 50 ng m\~^\ and rifampicin (Ciba Geigy), 100 ng m r ' tc ASI 9, othenvise 300 ng ml"'.

Preparation of total RNA from E. coli Thirty-millilitre culture samples were transferred to 40 ml Sorvall tubes containing 8 ml of sterile ice. The cells were harvested and resuspended in 200 pi of solution I (0.3 M sucrose, 0.01 M sodium acetate, pH 4.5). Two hundred microlitres of solution II (2% SDS, 0.01 M sodium acetate, pH 4.5) was added and the preparation was incubated at 65"C for 1.5 min. The preparation was extracted with phenol, and cooled in liquid nitrogen for 15 s. The phases were separated by centrifugation fcr 5 min and the phenol extraction repeated. Finally, the RNA was extracted with phenci/chlorcform at room temperature, precipitated with 3 vols of 96% alcohol plus 0.3 M sodium acetate, pH 4.7. The pellet was resuspended in 60 \i\ of TE and the amount of RNA was determined by measuring the OD260' The advantage of this method is its rapidity and its constant yield of RNA.

Northern transfer analysis The RNA samples (typically 20 ^g per sample) were fractionated on a denaturing 5.5% (7,5% for pndB transcript) poiyacryiamide gel (low bis-acrylamide) containing 8 M urea, and blotted onto a Zeta-probe nylon membrane (BioRad). After completion of the blotting, the membrane was prehybridized for 2 h before addition of the denatured single-stranded RNA probe. The filter was hybridized overnight and washed according to the manufacturer's prescription.

1972

A. K. Nielsen e\a\.

Acknowledgements The expert technical assistance of Marianne Hansen is greatly appreciated. We thank Poul Valentin-Hansen for helpful discussions. We also thank Per Hove-Andreasen and Marie Ohmann for help with the computer-fcldings of the antisense RNAs. This work was supported by a Grant from the Danish Centre for Microbiotogy.

References Akimoto, S., and Ohnishi, Y, (1982) R483 and F plasmid genes promoting RNA degradation: comparative restriction mapping, Microbioi Immunol26: 779-793, Akimotc. S., Ono, K,, Ono, T,, and Ohnishi, Y. (1986) Nucleotide sequence of the F plasmid gene srnB that promotes degradation of stable RNA in Escherichia coli FEMS Microbioi Letts 33:241 - 245. Akimoto, S,, Sakikawa, K.. Ono, T., and Ohnishi. Y. (1989) Transcriptional regulaticn of F plasmid gene srnS: rifampicin-promoted in vitro readthrough of a terminator in the leader region. Mol Microbioi Z: 787-796. Belascc, J.G., and Higgins, C.F. (1988) Mechanisms cf mRNA decay in bacteria: a perspective. Gene 72:15-23. Bolivar, F., Rodriquez, R.L., Greene, P.J., Betlach, ful.C, Heyneker, H.L., Boyer, HW., Crosa, J.H,, and Falkow, S. (1977) Construction and characterization of new cIcning vehicles. II. A multipurpose cIcning system. Gene 2; 9 5 113. Casadaban, M., and Cohen, S.N. (1980) Analysis of gene control signals in E. coli J Mol Biol 138:179-207. Gerdes, K., Larsen, J.E.L,, and Molin, S. (1985) Stable inheritance of plasmid RI requires two different loci, J Bacterioi 161:292-298. Gerdes, K., Rasmussen, P.B., and Molin, S. (1986a) Unique type of plasmid maintenance: postsegregational killing of plasmid-free cells. Proc Nati Acad Sci USA 83: 3116-3120. Gerdes, K,, Bech, F.W., Jorgensen, S.T., Lgbner-Olesen, A., Rasmussen, P.B,, Atlung, T., Karlstrom, O,, Mciin, S., and von Meyenburg, K, (1986b) Mechanism of postsegregalional killing by the hok gene product of the parS system of plasmid RI and its homology with the relF gene product cf the E, co/(re/S operon. EMBOJS: 2023-2029, Gerdes, K., Helin, K,. Christensen, 0,W,. and Labner-Olesen, A. (1988) Translational control and differential RNA decay are key elements regulating pcstsegregational expression of the killer protein encoded by the parB locus of plasmid R I . J Mo/S/o/203: 119-129, Gerdes, K., Thisted, T,, and Martinussen, J. (1990a) Mechanism cf post-segregational killing by the hok/sok system of plasmid R I : the so/cantisense RNA regulates the formation of a hok mRNA species ccrrelated with killing of plasmid free cells. Mol Microbioi A: 1807-1818, Gerdes, K., Poulsen, L,K,, Thisted, T,, Nielsen, A.K., Martinussen, J., and Hove-Andreasen, P. (1990b) The hok killer gene family in Gram-negative bacteria. New Biologist 2: 946-956. Iggo, R., Picksley, S., Southgate, J., McPheat, J., and Lane, D.P. (1990) Identificaticn of a putative RNA helicase in E. coli. NucI Acids Res 18: 5413-5417. tto, R,, and Ohnishi, Y. (1983) The roles of RNA polymerase and RNase I in stable RNA degradation in Escherichia coli carrying ^ e smB" gene. Biochim Biophys Acta 739: 27-34,

Kittle, J.D., Simons, R.W,, Lee, J., and Kleckner, N. (1989) Inserticn sequence IS 70 anti-sense pairing initiates by an interaction between the 5' end of the target RNA and a loop in the anti-sense RNA. JMolBiol2A0: 561-572. Larsen, J.E.L., Gerdes, K., Light, J., and Molin, S. (1984) Lowcopy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter. Gene 28: 45-54, Loh, S.M., Cram, D.S., and Skurray, RA. (1988) Nucleotide sequence and transcriptional analysis of a third function (Flm) involved in F-plasmid maintenance. Gene 66: 259268, Maniatis, T., Fritsch, E.F., and Sambrcok, J. (1982) Molecular Cloning. A Laboratory Manuai Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, Miller, J. (ed.) (1972) Experiments in Molecular Genetics. Cold Spring Harbor, New York: Cold Spring Harbor Labcratory Press, Ohnishi, Y. (1974) F factor promotes turnover of stable RNA in Escherichia coii. Science 187: 257-258. Ohnishi, Y., and Akimoto. S, (1980) l-like R-plasmids promote degradation cf stable ribonucteic acid in Escherichia coli. J Bacterioi-{44:833-835. Ohnishi. Y.. and Schiessinger, D. (1972) Total break-down of ribosomai and transfer RNA in a mutant of Escherichia coli Nature New Biol 238: 228-231, Ohnishi, Y.. Iguma. H., Ono, T., Nagaishi, H.. and Clark, A.J. (1977) Genetic mapping of the F plasmid gene that promotes degradation of stable ribonucieic acid in Escherichia coti J Bacterioi ^32: 784-789. Ohnishi, Y., One, T., Ito, M,, Akimoto, S. (1981) Cloning of a 1.1 kb fragment including srnS* gene in the F plasmid and isolation of an smB mutant. Microbioi Immunol 25: 12431254. One, K., Akimoto, S., and Ohnishi, Y. (1987) Nuclectide sequence of the pnd gene in plasmids R483 and role of the pnd gene product in plasmolysis. Microbioi Immunol 3 1 : 1071-1083. Pato, ML., Bennett, P.M., and von Meyenburg, K. (1973) Messenger ribonucieic acid synthesis and degradation in Escherichia coli during inhibition of translation, J Bacferiol 116:710-718. Pedersen. S,, Reeh, S., and Friesen, J, (1978) Functional mRNA half-lives in E. coli Mol Gen Genen66: 329-336. Persson, C , Wagner. E.G.H,, and Nordstrbm, K, (1988) Control of replication of plasmid R I : kinetics of in vitro interacticn between the antisense RNA, CopA, and its target, CopT. E/waO J7:3279-328B. Persson, C , Wagner, E.G.H.. and Ncrdstrbm, K. (1990) Control of replication of plasmid R I : Structures and sequences of the antisense RNA, CopA. required for its binding to the target RNA, CopT. EMBOJS: 3767-3775. Poulsen, L.K,. Larsen, N,W., Molin, S.. and Andersson, P. (1989) A family of genes encoding a cell-killing function may be conserved in all Gram-negative bacteria. Mol Microbioi 3: 1463-1472. Pculsen, L.K., Refn, A., Molin, S., and Andersson. P. (1991) The gef gene from E. coli is reguiated at the level of translaticn. Mol Microbioi 5: 1639-1648. Rasmussen, PB,, Gerdes, K., and Molin. S, (1987) Genetic analysis of the parS* Iccus cf plasmid R I . Mol Gen Genet 209: 122-128. Sakikawa. T.. Akimoto. S., and Ohnishi. Y. (1989) The pnd gene in E. coli plasmid RI 6: nucleotide sequence and gene

Antisense RNA-reguiated kilter genes srnB and pnd expression leading to Mg^* release and stable RNA degradation. Biochim Biophys Acta ^G07:158-166. Sekiguchi. M., and lida, S. (1967) Mutants of Escherichia coii permeable to actinomycin. Proc NatI Acad Sci USA 58: 2315-2320. Simons, R.W., and Kleckner, N. (1988) Biological regulation by antisense RNA in prokaryotes. Ann Rev Genet22: 567-600. Tomizawa, J. (1984) Control of ColEI replication: the process of binding of RNA I to the primer transcript. Celt 38: 8 6 1 870. Tomizawa, J., Itoh, T., Selzer, G., and Som, T. (1981) Inhibition of ColEI RNA primer formation by a plasmid specified small RNA. Proc NatI Acad Sci USA 78:1421-1425. von Gabain, A., Belasco, J.G., Schottel, J.L., Chang, A.C.Y,

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and Cohen, S.N. (1983) Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proc NatI Acad Sci USA 80:653-657. Wagner, E.G.H.. and Nordstrom, K. (1986) Structural analysis of an RNA molecule involved in replication control of plasmid H^. NucI Acids ResU: 2523-2538. Yanisch-Perron, C , Vieira, J., and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences ofthe M13mp18 and pUC19 vectors. Gene 33:103-109. Zuker, M., and Stiegler. P. (1981) Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. NucI Acids Res B: 133-148.