doi:10.1006/jmbi.2001.5274 available online at http://www.idealibrary.com on J. Mol. Biol. (2002) 315, 541±549

Characterization of the Small Antisense CI RNA thatRegulates Bacteriophage P4 Immunity

Francesca Forti, Ilaria Dragoni, Federica Briani, Gianni DehoÁand Daniela Ghisotti*

Dipartimento di Genetica e diBiologia dei microrganismiUniversitaÁ degli Studi diMilano, Via Celoria 2620133, Milano, Italy

E-mail address of the correspondDaniela.Ghisotti@unimi.it

0022-2836/02/040541±9 $35.00/0

In the immune state bacteriophage P4 prevents expression of the replica-tion functions by premature termination of transcription. A small RNA,the CI RNA, is the trans acting factor that regulates P4 immunity, bypairing to complementary target sequences and causing premature tran-scription termination. The CI RNA is matured by RNAse P and PNPasefrom the leader region of the same operon it regulates. In this work webetter characterize this molecule. CI RNA copy number was determinedto be around 500 molecules per lysogenic cell. By S1 mapping we de®nedthe 30-end at 8423(�1); thus CI RNA is 79(�1) nt long. The minimumregion for correct processing requires two bases upstream of the CI RNA50-end and the CCA sequence at the 30-end. Computer analysis by FOLDRNA of CI RNA sequence predicts a cloverleaf-like structure formed bya double-stranded stalk, a minor and a major stem loop, and a single-stranded bulge. We analysed several cI mutations, which fall either in thesingle or double-stranded CI RNA regions. Base substitutions in the mainloop and in the single-stranded bulge apparently did not change CI RNAstructure, but affected its activity by altering the complementarity withthe target sequences, whereas a mutation in the secondary stem had adisruptive effect on CI RNA secondary structure. The effects of this lattermutation were suppressed by a base substitution that restored the com-plementarity with the corresponding base in the stem. Base substitutionsin the main stem caused only local alterations in the secondary structureof CI. However, when the substitutions concerned either G8501 or itscomplementary base at the bottom of the stem, CI RNA was not correctlyprocessed.

# 2002 Academic Press

Keywords: transcription termination; RNA processing; RNAse P;ash mutants; PNPase

*Corresponding author

Introduction

Antisense RNAs regulate gene expression in avariety of bacterial systems and, in particular, havebeen commonly found among accessory geneticelements such as phages, plasmids and trans-posons.1 In most cases the antisense RNAs aresmall molecules transcribed from the same DNAregion as their target RNAs, but in opposite orien-tation, and have the potential to form one or morestem-loop structures. Bases in the loops are criticalfor the ef®ciency and speci®city of these regulationsystems, being involved in the establishment ofinitial interaction between the antisense and its tar-get RNA (kissing reaction2), whereas the stems are

ing author:

important not only to keep nucleotides involved inthe kissing reaction in single-stranded loops, butalso as stability determinants.3 Antisense RNAmodulation of gene expression is mostly post-tran-scriptional and often occurs through inhibition oftranslation initiation or by leading to a differentprocessing of the target RNA.4 ± 6 Premature ter-mination of target RNA transcription caused byantisense pairing has also been described.7

The lysogenic state of bacteriophage P4 is con-trolled by a small RNA, the CI RNA, that causespremature termination of lytic operon tran-scription.8 The cI locus, encoding CI RNA, islocated in the leader region of the lytic operon,transcribed from PLE (Figure 1). Downstream of cIis kil, the ®rst translated gene of the operon.9 Earlyafter P4 infection, transcription starting from PLE

# 2002 Academic Press

Figure 1. Map of the P4 left operon and immunityregion. (a) The map of nucleotides 4500 to 9500 of theP4 genome is shown according to the complete P4sequence (GenBank accession number X51522), and thetranscripts synthesized early after P4 infection are indi-cated under the map. Open boxes indicate genes; thetranscription start points at PLE and PLL are indicated bya bent arrow; ®lled triangles correspond to the termin-ation sites. (b) The map of nucleotides 8150 to 8800 ofthe P4 genome is shown and the immunity transcriptsare drawn under the map. The coordinates of seqA, seqBand seqC sequences, indicated by open arrows, are 8676-8640, 8479-8432, 8424-8402/8384-8362, respectively.

Figure 2. P4 CI RNA. Computer-predicted structuresof CI RNA molecules were obtained by the FOLDprogram.33 (a) Wild-type CI RNA. The minimumsequence necessary for CI RNA processing is shownand the 50 and 30-ends of the mature CI RNA moleculeare indicated by [. The cI and ash mutations used in thiswork are shown. (b) CI ash3 RNA.

542 Characterization of P4 CI RNA

produces 4.1, 1.3 and 0.3 kb transcripts(Figure 1(a)). At 20 minutes postinfection, the twoformer RNAs are no longer present, sincetranscription termination occurring in the ®rst 300nt of the left operon prevents expression of thedownstream lytic functions. Thus, only theshort ``immunity transcripts'' are synthesized(Figure 1(b)).

The CI RNA is cotranscribed with its targetRNA and derives from RNase P and PNPase-dependent maturation of the primary transcript9 ± 11

(Figure 1(b)). Complementarity between the seqBregion of CI and seqA/seqC sequences of the leadertranscript must be maintained in order to have ef®-cient transcription termination at three sitesmapped within the ®rst 400 nt of the operon (t1, t4

and timm), preventing in this way expression of thedownstream replication genes12 (Figure 1). Thissuggests that CI RNA can act as a pseudo-anti-sense RNA by pairing with its target sites andinducing transcription termination. Modulation ofprimary transcript secondary structure and controlof translation initiation of the kil gene are probablyinvolved in CI-dependent transcriptiontermination.12,13

Regulation mechanisms that show striking simi-larity with P4 lysogenization control have beendescribed for bacteriophages P1, P7, �R73 andN15. In all these systems, a pseudo-antisense RNAcauses premature termination of a lytic operontranscription by interacting with complementarysequences in the leader region of the nascent tran-

script. Moreover, all the regulatory RNAs share avery similar secondary structure and derive fromprocessing of their target transcript leaderregion.9,14 ± 16

The P4 CI RNA 50-end is processed by RNase P,9

the ribozyme necessary for tRNA 50-endmaturation.17 Preferred substrates for RNase P areRNA stems with a 30-CCA single-stranded tail.18

This structure is present in the CI RNA precursortranscript (Figure 2(a)). However, the size of the CIRNA estimated by evaluation of electrophoreticmobility (69 nt9), suggested 30-end trimming after50-end processing. This does not occur in C4 RNAof phage P1 and P714 or in CA of N15.16 Thus, wedecided to determine the 30-end of CI RNA in amore direct way.

In this work, by making use of cI mutations, weprobed the CI RNA molecule structure and de®nedthe minimal region necessary for CI RNA proces-sing.

Results and Discussion

Determination of CI RNA copy number in P4lysogenic cells

To determine the number of CI RNA moleculespresent in P4 lysogenic cells, we compared inNorthern blot the signal of CI RNA extracted fromC-1a(P4) and known amounts of in vitro syn-thesized CI RNA. By PhosphorImager densitome-try we determined the presence of about 500molecules per lysogenic cell (data not shown).Thus, a relatively high number of regulatory mol-

Characterization of P4 CI RNA 543

ecules are present in the immune state. This can bea consequence of the particular way P4 regulatesimmunity, by causing premature transcription ter-mination rather than preventing transcriptioninitiation. Indeed, the PLE promoter is active in thelysogenic state19 and a concentration of regulatoryCI molecules suf®cient to ef®ciently terminate tran-scription is necessary.

Identification of the CI RNA 30-end

Maturation of CI RNA by RNAse P creates its50-end at nucleotide 8501, as determined by primerextension.9 The apparent size of CI RNA was esti-mated to be about 70 nt long by denaturating poly-acrylamide gel electrophoresis, using RNAmarkers as size standards.9 However, subsequentexperiments indicated that migration of the CIRNA molecule was very sensitive to small differ-ences in electrophoretic conditions (data notshown) and a more direct approach was thusrequired to de®ne its 30-end.

We ®rst performed an S1 nuclease digestion pro-tection experiment using as a probe the 8493-8395P4 DNA fragment, 30-end-labelled at 8493, internalto cI. The protected fragments were separated on6 % polyacrylamide denaturing gel, together with aMaxam and Gilbert sequence of the same DNAfragment (Figure 3(a)). The 30-end protected frag-ments comigrated with two Cs at 8421-8422, notresolved in the sequence.

To independently determine the length of CIRNA, we performed an S1 protection experiment,using as probes different P4 DNA fragments,internally labelled with [a-32P]ATP, obtained byPCR ampli®cation. The length of the fragmentsprotected from S1 nuclease digestion by CI RNAwas assessed by electrophoresis (Figure 3(b)). Pro-tected fragments of the same length were obtainedwith 8537-8377, 8501-8377, and 8537-8422 P4regions, suggesting that CI RNA ends wereinternal to the 8501-8422 region. (In the conditionsused, electrophoretic migration of the protectedfragment was equivalent to an 81 or 82 nt longDNA molecule.) On the other side, protection ofthe 8537-8433 region originated a fragment about10 nt shorter. On this basis, the 30-end of the CIRNA can be mapped at 8423(�1), coinciding withthe CCA sequence. Thus, the length of CI RNA is79(�1) nt.

Mutations in the CI RNA molecule

Computer analysis by FOLD RNA of CI RNAsequence predicts a cloverleaf-like structure formedby a double-stranded stalk, a minor and a majorstem loop, and a single-stranded bulge(Figure 2(a)). To probe this structure in vivo, weanalysed the effects of several cI mutations, bothpreviously described and new in vitro-createdpoint mutations (see Figure 2), of different classes,either in single or double-stranded regions. Differ-ent phenotypic aspects that characterize P4 immu-

nity were considered for each cI mutation: (1)lysogenization ability was evaluated by plaquemorphology and lysogenization frequency. (2) P4immunity expressed by the mutant cI genes wasevaluated by measuring the ef®ciency of plating ofP4 cI405 on strains expressing the mutant cI genescloned on a plasmid. (3) Control of kil expression,which depends on P4 immunity,13 was determinedby the fraction of cells surviving either infectionwith P4 cI mutants or induction of kil from a resi-dent plasmid. (4) The ability of the mutant phagesto ef®ciently terminate transcription starting at PLE

19

was evaluated by Northern analysis of the lyticoperon transcription pro®le at different times uponP4 cI infection. (5) CI RNA production was ana-lysed by Northern blot. The results are summar-ized in Table 1.

Mutations in the bulge

This class is the most abundant among P4 cImutants obtained in vivo. Seven out of eight basesof the bulge are complementary to both seqA andseqC. We analysed base substitutions in differentpositions. Some mutations had been isolated pre-viously in vivo: cI40520 (C8446U), ash821 (U8447C),ash921 (U8448A); others were created by site-directed mutagenesis: cI444 (C8446G) and cI446(C8444G). Furthermore, we analysed the singlebase insertion ash1021 (�C8441) that increases thebulge length to 9 nt (Figure 2(a)).

Base substitutions in any cytosine of the bulge(cI405, cI444 or cI446) caused the most drasticeffects: clear plaques, inability to lysogenize,absence of immunity to P4 cI405 superinfection,loss of kil expression control and of immunity-induced transcription termination. The two otherbase substitutions in the bulge that substitute twodifferent U (ash8 and ash9) and the insertion ash10had a less severe effect: all phages presented aclear plaque morphology and lysogenizationability was reduced (ash10) or absent (ash8 andash9); moreover, transcription termination from PLE

was relatively inef®cient, since the 4.1 and 1.3 kblong transcripts were still present 20 minutes, butwere not observed 60 minutes after infection. How-ever, all three phages were still able to control kilexpression, since a high fraction of C-1a cells sur-vived infection. (The surviving colonies were plas-mid P4 carriers; data not shown.) Thus, it appearsthat the delayed transcription termination observedin P4 ash8, ash9 and ash10 impairs immunity estab-lishment. However, phenomena such as cell killing,which require prolonged expression of kil, can beprevented.

Computer-predicted structures for all the RNAmolecules with single base substitutions were notaltered compared to wild-type CI, and the CI RNAproduced appeared normal by Northern blotanalysis. Accordingly, CI RNA produced by P4cI405 was correctly processed at the 50 and 30-ends(Figure 3(a) and (c)). The ash10 base insertion, forwhich a CI RNA structure with an increased bulge

Figure 3. Determination of CIRNA ends. (a) and (b) Determi-nation of CI RNA 30 by S1 nucleaseprotection. (a) The 8493-8395 P4DNA fragment obtained asdescribed in Materials andMethods, labelled at 8493, washybridized with RNA extractedfrom cultures of C-1a (ÿ), C-1a/pGM80 (cI�), C-1a/pGM569 (ash3),C-1a/pGM570 (ash29) and C-1ainfected with P4cI405 (cI405), anddigested with 263 units (�), 526units (��) of S1 nuclease or non-digested (ÿ). The Maxam-Gilbert Gsequence reaction of the DNA frag-ment was run in the same gel (G).The bases of the complementarystrand are indicated. The double C,corresponding to the 30-end, is indi-cated by an arrow. (b) The DNAfragments were synthesized byPCR ampli®cation in the presenceof [a-32P]dATP, as indicated inMaterials and Methods. The coordi-nates of the P4 regions covered bythe fragments are indicated on top.After hybridization with RNAextracted from C-1a/pGM80, theDNA fragments were digested with526 units of S1 nuclease (��) ornon-digested (ÿ) and separated on6 % polyacrylamide denaturing gelelectrophoresis. A DNA sequencewas run in the same gel as a mol-ecular mass marker. (c) Determi-nation of CI RNA 50-end by primerextension. RNA was extracted fromC-1a infected with P4 wild-type(cI�), cI405 (cI405), ash29 (ash29),ash3 (ash3) and from C-1a carryingthe plasmids indicated on top ofthe lanes. Primer extension wasperformed as described inMaterials and Methods. Thesequencing reactions were obtainedusing the same primer and plasmidpGM228 (sequence of the templatestrand). Position 8501 identi®es theC base that is complementary to

the G at the 50-end of the CI RNA. The protected bands corresponding to transcription initiation at the vector placpromoter are indicated on the left.

544 Characterization of P4 CI RNA

(�1) was predicted, produced a CI RNA moleculeone nucleotide longer than wild-type CI (data notshown).

These data suggest that base substitutions in thesingle-stranded bulge do not prevent CI RNA pro-duction and/or maturation; rather, such mutationsdirectly alter the active site of the molecule.Indeed, the most drastic effects were observedwith mutations that alter the two cytidine bases inthe central region of the bulge, whose role is prob-ably more relevant in creating the contact with thetarget RNAs. Interestingly, cI405 changes a cyti-

dine base to uracil, thus pairing with the guaninebase in either seqA or seqC should be possible.However, the spontaneous cI405 mutant is one ofthe strongest ever isolated, suggesting that theweak U-G interaction cannot substitute for thestrong C-G interaction.

Mutations in the major loop

No mutations had been previously found in vivoin this single-stranded region, complementary tothe seqA and seqC targets of CI RNA. Thus, we cre-

Table 1. Phenotypic analysis of the P4 cI mutations

Lysogens/ Efficiency of Control of kil expression Transcription terminationg

Mutationa PositionbPlaque

morphologycinfected cellsc

(%)plating of P4

cI405d Survivorse (%) Growthf 10 20 60 CI RNAh

cI � - - tu 33 <10ÿ6 83 � � ÿ ÿ �cI405 C8446T Bulge cl <0.01 1 <1 ÿ � � � �cI444 C8444G Bulge cl <0.01 1 <1 ÿ � � � �cI446 C8446G Bulge cl <0.01 1 <1 ÿ � � � �ash8 T8447C Bulge cl <0.01 nti 68 nti � � ÿ �ash9 T8448A Bulge cl <0.01 nti 80 nti � � ÿ �ash10 �C8441 Bulge cl 5� <10ÿ4k 94 nti � � ÿ � (�1)cI6063 G8460C/C8463G Major loop cl <0.01 1 <1 ÿ nti nti nti �cI478j C8478T Minor loop nth nth <10ÿ6 nti � nti nti nti �ash3 C8474A Minor stem cl <0.01 1 <1 ÿ � � � �ash3 cI483 C8474A/G8483T Minor stem tu 9 <10ÿ6 nti � � ÿ ÿ �ash23 T8434C Major stem int 17� <10ÿ4k 54 nti � ÿ/� ÿ �ash29 G8433A Major stem cl 19� <10ÿ6 63 nti � � ÿ/� �ash29 cI92j G8433A/C8494T Major stem nti nti <10ÿ6 nti � nti nti nti �cI494j C8494G Major stem nti nti 10ÿ2 nti ÿ nti nti nti �cI501j G8501A Major stem nti nti <10ÿ6 nti nti nti nti nti ÿcI426* C8426A Major stem nti nti <10ÿ6 nti nti nti nti nti ÿ

a The mutations in the cI gene carried by phage P4 and/or cloned in a plasmid are indicated, with the coordinate and the base substitution.b Position in the CI RNA computer-predicted structure (Figure 2(a)).c Plaque morphology and lysogenization were determined upon infection of C-5205 and C-117, respectively. Turbid (tu), clear (cl), intermediate (int) plaque morphology. (�) the amount of

phage released by the lysogenic strains was about 100-fold increased relative to P4� release.d Expression of P4 immunity was evaluated by measuring the ef®ciency of plating (eop) of P4 cI405 on C-5510 carrying the plasmids in which the cI gene is cloned downstream of plac.e Control of kil expression was evaluated by measuring the fraction of C-1a cells surviving infection with P4 mutants (multiplicity of infection ca 10).f Control of kil expression was evaluated by determining growth ability of C-1a carrying plasmids in which the kil gene was cloned downstream of plac (P4 8657-7629 fragments of the different

cI mutants). Single colonies were replicated in the presence of IPTG (40 mg/ml): (�) growth; (ÿ) absence of growth.g Immunity transcription termination was determined by Northern blot analysis of the transcripts synthesized upon C-1a infection with the mutant phages: the presence (�) or absence (ÿ) of

the 4.1 and 1.3 kb transcripts was monitored at 10, 20 and 60 minutes after infection. Persistence of the 4.1 and 1.3 kb transcripts at 20 and 60 minutes after infection indicated a defect in tran-scription termination.

h RNA was extracted either from C-1a infected cells (60 minutes upon infection) or from C-1a transformed with different plasmids and the presence of CI RNA was detected by Northern blotin 6 % polyacrylamide denaturing gel, using the SeqB oligonucleotide as a probe.

i Not tested.j These mutations have not been transferred into the phage.k eop of P4 cI405 on C-117(P4 ash10) and C-117(P4 ash23) lysogenic strains.

Chara

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P4

CI

RN

A545

546 Characterization of P4 CI RNA

ated a double base substitution, cI6063 (G8460Cand C8463G), by site-directed mutagenesis(Figure 2(a)). Also in this case the computer-pre-dicted structure of CI did not change, and CI RNAwas normally produced. As a consequence of themutations a severe phenotypic effect was observed,with complete loss of immunity control. Thus, thebases in the main loop appear to be involved in CIRNA activity. These data con®rm the hypothesisthat the bases in the bulge and in the main loopare directly involved in the ``kissing''2 betweenseqB and the seqA and seqC targets.10

Mutation in the minor loop

The minor loop does not exhibit complementar-ity with either seqA or seqC, and no spontaneousmutants in this region had been previously found.However, the four bases of the minor loop are con-served in P1 and P7 C4, N15 CA and �R73 CIRNAs (consensus sequence UYCG), as well as theC:G base-pair delimiting the loop.14,16 Thissequence constitutes a tetraloop structure, fre-quently found in rRNA hairpin loops,22 that mightincrease RNA stability. We created a C8478T sub-stitution in this region (cI478) in the cloned cI gene(Figure 2(a)). Both computer-predicted CI structureand CI RNA production appeared normal; further-more, this mutation neither affected P4 immunitynor eliminated control on kil expression. Thus, thecI478 mutation did not appear to have any sub-stantial effect on CI RNA production and activity,at least when CI is expressed from a plasmid. Onecannot exclude that different base changes in thetetraloop sequence could alter CI RNA stabilityand/or activity, and might affect P4 lysogenizationability.

Mutations in the minor stem

The in vivo isolated ash3 mutation21 is a basesubstitution in a double-stranded region (C8474A)that eliminates complementarity with the corre-sponding base in the minor stem. The secondarystructure of the CI RNA predicted by the FOLDprogram is completely altered, the major stem isstill present, but the bulge and the minor stem-loops are not formed (Figure 2(b)). The ash3mutant produced an apparently normal-sized CIRNA and both the 50 and 30-ends were correctlyprocessed (Figure 3(a) and (c)). This con®rms thatRNase P processing of CI RNA depends mainly onthe RNA structure at the base of the stalk. On theother hand, drastic phenotypic changes werecaused by ash3: clear plaque morphology, inabilityto lysogenize, absence of immunity to superinfec-tion, loss of transcription termination and kilexpression control.

We constructed by site-directed mutagenesis adouble mutant carrying ash3 and the cI483mutation (G8483T); the second is a suppressormutation that restores complementarity betweenthe bases of the secondary stem (Figure 2(a)).

Computer-predicted analysis of the CI RNAmolecule produced by the ash3 cI483 doublemutant was not distinguishable from wild-type CI.Furthermore, in vivo all the phenotypic alterationscaused by ash3 were suppressed. The only minordifference observed in comparison to P4 wild-typewas a slight reduction in lysogenization ef®ciency(9 % compared to 33 %). These data suggest thatthe main defect caused by the ash3 mutation con-cerns CI RNA secondary structure.

Mutations in the major stem

Two spontaneous mutations in the main stem,ash2321 (T8434C) and ash299 (G8433A) and thein vitro-created cI494 mutation (C8494G), do notchange the overall conformation of CI RNA andsimply create a mispaired bubble in the middle ofthe major stem (data not shown). All the mutantsproduced CI RNA and the CI RNA molecule pro-duced by ash29 had both 50 and 30-ends correctlyprocessed (Figure 3(a) and (c)). Effects on P4immunity caused by each of the three mutationswere relatively mild (Table 1). Both P4 ash23 andP4 ash29 were still able to lysogenize the Escherichiacoli host, although a greater amount of phage wasreleased by the lysogens, suggesting a more fre-quent induction of the prophages. Moreover, bothphages were able to control kil expression. P4 ash23presented a modest defect in transcription termin-ation: the 4.1 and 1.3 kb transcripts were stillobserved 20 minutes after infection, but not at latertimes, whereas P4 ash29 more drastically preventedtranscription termination. Expression of cI494 froma plasmid conferred partial immunity to the cell,insuf®cient, however, to control kil expression.

We also constructed a suppressor mutation ofash29 that restored complementarity in the double-stranded stem (cI92; C8494T; Figure 2(a)). Noapparent defects in immunity expression wereobserved in the ash29 cI92 double mutant.

Two single mutations that alter the bases at theend of the main stem were created: cI501 (G8501A)and cI426 (C8426A). Both prevented pairing of the50-end of the mature CI RNA to the opposite basein the stem. These mutations prevented CI RNAproduction. A longer molecule, whose 50-end wassix bases upstream of 8501, was produced by bothcI501 and cI426 (data not shown), indicating thatcorrect pairing of G:C at the bottom of the majorstem might be essential for correct processing ofthe CI RNA by RNAse P. (It should be noted that,although incorrectly processed, the RNA moleculesproduced by cI501 and cI426 mutant genes werestill able to confer P4 immunity to the cell; Table 1.)An identical result was observed in N15 by chan-ging the G at the 50-end of CA RNA.16 It cannot beexcluded that the sequence itself covers a relevantrole in RNase P processing. Indeed, the ÿ2 to �5sequence at the 50-end of P4 CI is highly conservedin CI-like molecules (the consensus sequence isAUGGURG13,16).

Characterization of P4 CI RNA 547

Identification of the minimum region requiredfor CI RNA production

CI RNA is produced by processing of longertranscripts.9 To de®ne the minimum region suf®-cient for CI RNA production, we cloned several P4regions, all including the cI locus, and testedwhether the wild-type CI RNA could be produced.The P4 regions cloned in the plasmids and North-ern blot analysis of the RNAs produced are shownin Figure 4.

Plasmid pGM627, in which the P4 region clonedstarts two bases upstream of 8501 (the 50-end of themature CI RNA9), produced a CI RNA molecule ofthe same length as CI RNA produced by P4(Figure 4(b)) and with the correct 50-end(Figure 3(c)). A slight reduction in the amount ofCI RNA produced by pGM627 in comparison to CIRNA produced by plasmids carrying a moreextended region (pGM282, pGM576 and pGM577)suggests that processing might be less ef®cient inthe former.

When the cloned P4 region started exactly at8501 (pGM626), the CI RNA molecule appearedlonger than wild-type CI (Figure 4(b)) carrying sixbases of vector sequence at its 50-end as determinedby primer extension (Figure 3(c)). Thus, one or two

Figure 4. CI RNA production and immunity expression byried by the plasmids and immunity expression. The P4 regionates and by grey bars below the map. Coordinate �1, in ththe mature CI RNA molecule. Expression of P4 immunity w(eop) in C-5510 carrying the different plasmids: (�) high leve(eop � 10ÿ2), (ÿ) absence of immunity (eop � 1). (b) NortheThe RNAs extracted from C-1a carrying the indicated plasmielectrophoresis, blotted on nylon ®lter and hybridized withWild-type CI RNA (®rst lane) was run in the same gel as a c

bases upstream of 8501 appear to be required forcorrect and ef®cient processing of the 50-end byRNase P. Although the pGM626 CI RNA differedfrom the wild-type molecule, it was still able toconfer P4 immunity to the cell (see Figure 4(a)).

Deletion of ®ve bases at the 50-end (pGM297)prevented processing of CI RNA. It should benoted that a low level of P4 immunity was stillexpressed by this plasmid, suggesting that RNAmolecules that cover the cI locus, although not cor-rectly processed, are still partially functional.pGM82, in which most of the cI gene is deleted,completely lost CI RNA production and P4 immu-nity expression.

The correct processing of the CI RNA 30-endrequired the terminal CCA: pGM577 that includesthe CCA sequence produced CI RNA, whereaspGM578 that lacks CCA was unable to synthesizeit. Also in this case, a low level of immunity wasexpressed by the latter strain.

Our analysis indicates that the minimal require-ments of the P4 CI RNA precursor are the AUGsequence (ÿ2 to �1) at the bottom of the majorstem, with G paired to C in the opposite strand,and a protruding CCA sequence at the 30-end.These results are in agreement with the relevant

plasmids carrying P4 cloned regions. (a) P4 regions car-ns cloned in the plasmids are indicated by the P4 coordi-e scale above the map, corresponds to 8501, the 50-end ofas evaluated by measuring P4 cI405 ef®ciency of platingl of immunity (eop <10ÿ6), (�/ÿ) low level of immunityrn blot analysis of CI RNA produced by the plasmids.ds were separated by denaturing 6 % polyacrylamide gelriboprobe SeqB, as indicated in Materials and Methods.ontrol.

548 Characterization of P4 CI RNA

features (ÿ2 to �1 region at the 50-end and RCCsequence at the 30-end) necessary for correct andef®cient tRNA precursors processing by E. coliRNase P.23

Materials and Methods

Bacterial strains, bacteriophages and plasmids

The bacterial strains used were the Escherichia coli Cstrains C-1a (prototrophic),24 the P2 lysogenic strains C-117,25 C-520526 and C-5510 (pcnB, recA, lysogenic for P2lg).27 The phages used were P4,27 P4 ash3, P4 ash8, P4ash9, P4 ash10, P4 ash23,21 P4 ash29,9 P4 cI405,20 P4 cI444,P4 cI446, P4 cI6063 and P4 ash3 cI483 (this work).

The plasmids used in this work are pUC8,28 pUC18 orpUC1929 derivatives carrying the P4 DNA regions indi-cated in parenthesis, downstream of the plac promoter:pGM80 (P4� 8589-8420), pGM91 (cI405 8624-8420),pGM321 (P4� 8659-7628), pGM360 (P4� 8776-8209),pGM379 (cI501 8776-8209), pGM380 (cI426 8776-8209),pGM561 (cI478 8657-8420), pGM562 (cI6063 8657-8420),pGM563 (ash3cI483 8657-8420), pGM566 (ash29cI92 8657-8420). Other pUC8 derivatives are indicated in Figure 4.pGM228 is a pKO1 derivative30 carrying the P4 � 8775-8130 region.

Construction of cI mutations

The cI mutations were obtained by PCR ampli®cationof the 8367-8622 P4 DNA region with mutated oligonu-cleotides by using the ``overlap extension'' technique.31

The mutant fragments were cloned in pGM321, replacingthe wild-type MluI 8622-BglI 8367 P4 DNA region. TheMluI 8622-Tth111I 7776 fragments from the resultingplasmids were ligated with the Tth111I 7776-0-MluI 8622P4 fragment and strain C-5205 transfected. The presenceof the mutation in phage DNA was assessed by sequen-cing the cI region.

Northern blot hybridization and primer extension

RNA was extracted from E. coli and from P4-infectedcells, fractionated on a 6 % (w/v) polyacrylamide-ureadenaturing gel and transferred to a Hybond N ®ltermembrane (Amersham) as described previously.19 The32P-labelled riboprobe SeqB was prepared by SP6 poly-merase transcription of the 8418-8470 P4 DNA region.The SeqB oligonucleotide 50-GGTGAGAA-TACCGGCTTC-30 (P4 coordinates: 8441-8458) was end-labelled with T4 kinase. Hybridization was performed aspreviously described.19 The 50-ends of CI RNAs wereidenti®ed by primer extension analysis, using the SeqBoligonucleotide.9

S1 protection experiments

The 8493-8395 P4 DNA fragment, obtained by PCRampli®cation and digestion with SmaI and DdeI, was 50-end-labelled at 8493 by ®lling-in the DdeI overhangingend by the Klenow fragment of E. coli DNA polymeraseI. The 8537-8377, 8501-8377, 8537-8422, 8537-8433 frag-ments were obtained and internally labelled by PCRampli®cation in the presence of [a-32P]dATP. Thelabelled fragments were puri®ed by electroelution,denatured, hybridized with RNA and treated with S1

nuclease as previously described.32 The S1 digests wererun on a denaturing 6 % polyacrylamide gel.

Acknowledgements

We thank E. Six for kindly providing P4 ash mutantsand A.M. Barbieri for technical support. F.B. was a reci-pient of a fellowship from UniversitaÁ degli Studi di Mila-no, Milan, Italy. This work was supported by grant no.99.02493.CT04 of the Consiglio Nazionale delle Ricerche,Rome, Italy and by grants from the Ministero dell'Uni-versitaÁ e della Ricerca Scienti®ca e Tecnologica, Rome,Italy.

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Edited by M. Gottesman

(Received 31 July 2001; received in revised form 22 October 2001; accepted 15 November 2001)