0950382X9100011C

Molecular Microbiology (1991) 5(1), 77-87

Repression and derepression of conjugation of piasmid R1 by wild-type and mutated f/nP antisense RNA G. Koraimann, C. Koraimann, V. Koronakis,^ S. Schlager and G. Hogenauer* Institut fur f\Aikrobiologie, Karl-Franzens-Universitat Graz, Universitatspiatz 2, A-8010 Graz, Austria. Summary The finP gene of piasmid R1 is located between the genes traM and traJ, partially overlapping the first few nucleotides of the latter. It codes for a repressor of the conjugation system. The product of this gene is a small RNA of 72 nucleotides and, because it is transcribed from the opposite DNA strand, it is complementary to the 5' non-translated sequences, the ribosome-binding site, and the first two codons of traJ mRNA. The finP transcript is present in much higher concentrations in R1 than in R1-19 containing cells, the latter being a derepressed mutant of the former. A synthetic finP gene expressed from a synthetic lambda PL promoter markedly reduced the conjugation frequency of pDB12, a multicopy derivative of Rl-19. Mutagenesis of finP showed that only finP loop II mutants have lost the ability to repress conjugation of R1-19 in trans. They are also the only ones which derepress conjugal DNA transfer of R1, probably by competing for the finO product, a molecule needed as corepressor for maximal activity. Mutations interrupting potential open reading frames of finP do not abolish finP repressor activity. Hence finP acts as an antisense RNA blocking the expression of the traJ gene by interacting with traJ mRNA through loop II.

Introduction The conjugative resistance piasmid R1 belongs to the incompatibility olass InoFII and is thus related to piasmid Rl 00 or to the F factor of Escherichia coli. The majority of the genes necessary for the ability to conjugate are arranged in the transfer (tra) operon and are positively regutated by the product of the traJ gene (Willetts, 1977). rraJexpression, in turn, is controlled at the post-transcriptional level by two negative elements, finP and finO. The finP genes are plasmid-specific vi/hereas the finO gene is Received 4 June, 1990; revised 6 August, 1990. tPresent address: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK, *For con-espondence. Tel. (316) 380 5620; Fax (316)382130.

exchangeable among F-)ike plasmids. finP is believed to code for a small antisense RNA, and the finO product is required for full repressor activity. The finO gene contains an open reading frame (ORF) ooding for a polypeptide of 21 kD which Is interrupted by an IS3 sequence in the F piasmid {Cheah and Skurray, 1986; Yoshioka et ai, 1987), As a result, the F piasmid conjugation is derepressed, meaning that all bacterial oells harbouring the F piasmid are conjugation-proficient, whereas conjugation of Rl or R100 is repressed, which means that only about 0.1 % of the cells containing one or the other of these plasmids promote piasmid DNA transfer into recipient cells. However, it is not clear whether the active finO product is a protein or a third RNA species involved in control of fraJ expression (Dempsey, 1987). A directly measurable effect of FinO is the increase of the intracellular concentration of finP RNA oy a mechanism which does not affect transcription (Mullineaux and Willetts, 1985) but rather involves stabilization of the latter (Frost efa/., 1989). The finP product seems to be the primary factor in the regulatory events because of its ability to bind to the 5'-end of the rraJ messenger RNA including the ribosomebinding site. This pairing theoretically prevents the access of ribosomes and, as a consequence, inhibits traJ mRNA translation. The existence of this mechanism of downregulation of gene expression by an antisense RNA was recently experimentally confirmed in the IS 70 regulation of transposase expression {Ma and Simons, 1990). Although it is likely that the finP product is an RNA this assumption has never been formally proven. By studying the ftnP gene in the Rl-system we could show that the corresponding DNA region of ptasmid R1 codes for an RNA which suffices to strongly repress conjugation of piasmid Rl-19 /nVrans. Rl-19 is a laboratory mutant of R1 {Meynell and Datta, 1967) which is derepressed in its conjugation like the F piasmid. Moreover, mutagenesis experiments underscore the importance of loop II of the predicted secondary structure of finP RNA for the function of FinP and exclude the possibility that the repressor acts as a polypeptide.

Results Location and size of finP RNA The RNA transcript of the finP gene was detected by three independent methods, i.e. by probing of DNA fragments

78

G. Koraimann et al.

£"07 RV

Xi>al

EcoRl

TRAM

THflfl STOP

10

20

3D

40

RNfl STOP i

ftTTT&CTTCAftftTftftCft&Gfcn T I & l T C T G 3'

-

flTTTTTTCTI

AATTTICAflC

TAAACGAAGT

TTATTGfCCT

SO

6C

&TTCMi,TTT&

AAAACIAGAC

CAAGTTAAAC TRAJ

l

R^A

START

70 80 90 100 110 120 ATGACAATIA flCCGCATAAA GGTTATATTA AITACGTGGT TAATGCCACG TTAAAAAfTG TACTGirAAT IGGCGTATTT CCAATATAAT FAATGCACCA ATTACGOTGC AATTTIIAAC

liO 180 150 160 170 ftAACTGAAAA TC&CCGAT&C ftOGGAGGTCl GAACICCLTG CATCGfiCTgi TTTGACTTTT AGCGGCTACG TCCCTCCAGA CrrGflGGGACGlAGCTGACA GGTATCUAG f

t

TRA..

STOP

*•

*

START

190 1200 210 220 250 CTTAACGGtftG GTICCTAIGT GTGCGCTGGA fCGTAGAGAA AGGCCACTTA GAAI fGCCTC LAAGGATACA CACGCGACCT AGCATCTCTT TCCGGlGAAi FINP

START

with metabolically labelled RNA, by reverse transcription using specific primers, and by RNA-RNA hybrid protection experiments. For the Southern hybridization experiment either R I - or R1-19-containing £ coti cells were metabolically ^^Plabelled and the total RNA was extracted. The 4S RNA was subsequently separated from other RNA species and used to probe various DNA fragments around the expected location of the finPgene. Only those DNA segments which contained the intercistronic region between genes traM and trad, as in pSFI211 and in pSFI218 {Koronakis and Hogenauer, 1986), showed clear signals. The genetic map of the insert of pSFI211 and the sequence of the intercistronic region is shown in Fig. 1. pSFI216, which lacks this region but contains most of traM, shows no signal. pSFI218 carries the HincW fragment, and pSFI216 the HincW (2)/EcoRI fragment. RNA from R1-19-containing cells gave a much weaker signal with the same DNA fragments (Fig. 2). Hence the gene for the RNA was localized at a DNA segment between the Pst\ (2) site in traJ and the EcoRV site in traM. The length of the finP RNA was measured by hybridization of /n-v'iro-synthesized ^^P-labelled RNA, which is complementary to the finP RNA, and digesting the resulting RNA-RNA hybrid with a mixture of ribonucleases A and T1. The source of finP RNA for this experiment was an

240 ACAGICAATC - 3' IGKAGTIAQ - 5' -35

Fig. 1. Genetic and physical map ot the 5.3kb EcoFH/Pst\ |1) DNA fragment ot Rl contained witfiin pSFI211. Only relevant restriction sites are shown. The sequence ot the intercistronic region between traM and traJ plus a short stretch ot the fraJ coding region is shown in detail. The DNA sequence and the genetic signais lor traM and traJ are from Koronakis and Hogenauer (1986). The transcription signals tor finP were determined in this study. The sequence of the oligonucleotide used in the reverse transcnption of firjP RNA is boxed. Inverted repeats are indicated by arrows.

extract from E. coli cells containing the recombinant plasmid pSFI210 with the 7.7 kb Eco Rl fragment E (Ostermann et a/., 1984) from Rl as an insert. Three different RNA probes were made in vitro using the vector pSP64 and the SP6 RNA polymerase on the following templates: (i) an XtJal/EcoRV fragment, containing only part of gene 1-

(O

B

Fig. 2, Southern blot: ''^P-labelled 4S RNA trom Rl (A) or R1-19 (B) containing E. coli cells were hybridized to appropriately cut, electrophoretically separated and immobilized plasmid DNA tragments ct the indicated subclones. An autoradiogram ot this taiot is shown.

finP RNA from R1 79 A B C 217 201 190 160 160 U7

122 110

90

76

of the finP RNA. Electrophoresis of the remaining RNA on a denaturing polyacrylamide gel allowed its size determination. Only RNA synthesized on the latter two templates was partially protected. As can be seen from Fig. 3 the largest well represented RNA band measures 72 nucleotides {nt). The 5'-end of the finP RNA was determined by reverse transcription of oligonucleotide primers. One oligonucleotide, which appears boxed in Fig. 1, yielded detectable products with RNA extracted from R1- and R1-19-containing cells as well as from cells containing subclones pSFI210 and pSF1211. Electrophoresis and size measurement (Fig. 4) allowed us to assign the RNA start of the finP gene to the nucleotides denoted by arrows in Fig. 1. The transcriptional start appears to be heterogenous, the major position being the A at nucleotide number 202. If the experimentally determined length of 72 nt is added one would assume that the 3'-terminus of the finP RNA is located at position 131. However, since it is known from the literature {Platt and Bear, 1983) that transcripts after rho-independent terminators end with a series of undine residues, the transcription stop in Fig. 1 was assumed to occur at position 127. If termination indeed occurs at this position, the existence of a 72 nt RNA can only be explained if four nucleotides are removed from the 3'-terminus. It is also possible that longer primary transcripts are converted to the 72-nt molecule. The varying band intensities reflect the different finP RNA concentrations of R l - and R1-19-containing cells. The result supports the

E

Fig. 3. RNA-RNA protection experiment: in vitro synthesized '^Piabeiied RNA compiementary to finP RNA was hybridized to totai RNA extracted trom pSFi210 containing E. coli celis. The resuiting hybnds were digested with a mixture ot RNase A and T l . and the protected RNA fragments were subsequentiy separated on a denaturing poiyacryiamide gei. An autoradiogram of the gei is shown. The RNA probes were transcribed from Ihe foiiowing templates. A, Xbal/EcoRy fragment; B, Xbal/Pstl (2) fragment; and C, EcoRW/Pstl (2) fragment.

traM and lacking the intercistronic region between traM and traJ; (ii) an Xba\/Pst\ (2) fragment, containing the whole traM gene and part of the traJ gene; (iii) an EcoW/ Pst[ (2) fragment which covers the 3'-end of the traM gene, the intercistronic region and part of the traJ gene. The RNAse treatment of the RNA-RNA hybrids removed the single-stranded non-annealed ends of the radioactive RNA molecules, which were reduced in size to the length

Q:

o

^

W

CM

IT

t^

Q:

34 nt

26 nt

Fig. 4. Reverse transcription ot total RNA isolated from E. coii celis harbouring different plasmids as indicated. The reverse transcripts were eiectrophoreticaily separated on a denaturing poiyacryiamide gel. The autoradiogram of the gei is shown.

G. Koraimann et al.

pALlS

ori

BamHI

Hlndlll

/

A, PL Promoter Oligo 1 Oligo 2

/m

p

Oligo 3 Oligo 4

\ 1

e-GATCCAfiATA ACCATaGCG 6T6ATAAATT ATaCTGGCG GTGnSACAT AAATACMCT 3-GTCTAT TG6TAGACGC CAaATHAA TAGAGACCGC aCAACTGTA rTTATGGTGA GGC6GTGATA aGAGCAC CCGCCAQAT GAaCGTGTT

GtGTA TCCTTSGAGG CAATTCCTAA GATACCTGTC M I

ITCGATGCAGfi flASTTCAfiAC CTCCCTfiCAT C66C6ATmi CTGa y ASCTACGTCC CTCAASiaS 6AGG6ACSTA 6CC6CTAAAA G B" Fig. 5. The conslruction of pALi3. pACVCl77 was linearized wilh SamHI and Ps(l. The reassocialed and subsequently ligated oligonucleotides were cloned into the cut vector plasmid to give pALI 3. The boxed sequence denotes the sequence of the finP gene. The transcription start site is marked,

data obtained with the /n-wVo-labelled RNA shown in Fig. 2. Cells harbouring R1 contain higher measurable concentrations of fmP RNA than cells carrying R1-19. Using reverse transcription, the R1-19-containing cells did not show such low FinP concentrations as the direct probing with the metabolically labelled RNA indicated. We did not investigate further as to whether this difference is due to the methods applied or whether it reflects fluctuations in the cellular content due to different growth characteristics. The reason for the differences in the pattern of the subclones is unknown. A second oligonucleotide spanning the sequence from position 113-132 gave no reverse transcript, which means that this oligonucleotide lies beyond the 3'-end of the linP RNA molecule. This is in accord with the results obtained (described above) using length measurement by the RNA-RNA protection.

shown in Fig. 5. The recombinant plasmid pAL13 was introduced into a strain with a thermosensitive lambda repressor. At the non-permissive temperature significant amounts of transcript were formed, as was shown by Northern hybridization using a ^^P-labelled 4.7kb BglW/ EcoRI fragment, obtained from pSFI210. as a probe. No transcript could be detected at the permissive temperature (Fig. 6). However, under the control of the active lambda promoter, the synthetic gene produced longer transcripts than expected. Two transcripts of c. 400 and 570 nt were observed. Both RNAs are considerably longer than the ftnP RNA formed in its natural context. However, this larger RNA was active as a repressor when tested in a conjugation assay. This assay measures the conjugation frequency of pDB12, a deletion variant and a copy mutant of R1 -19, which lacks all the antibiotic resistance determinants except that for chloramphenicol (Blohm, 1979), in the presence o1 the recombinant plasmid pAL13 or pACYC177 as a control plasmid. The latter showed no effect on the conjugation frequency of pDB12. The results with pAL13 are shown in Table 1. Basically conjugation was repressed only when the cells were grown at the non-permissive temperature (42''C) of the lambda repressor. Conjugation at 42''C with previous growth of the cells at the permissive temperature had no effect on the conjugation system. The conjugation frequency was lowered to c. 6% of the non-repressed system.

a. o » n

kb

i

9 (10

80

a1

1o

u 1 ; 1

5f

1

Cl

I

4

1

a

0,60,40.3-

Construction and expression of a synthetici\nP gene The subclone pSFi211, but not pSFI216, repressed conjugation of Rl -19 by approximately IQ-fold when present in the same cell (Ostermann et al., 1984). The former plasmid contains, and the latter lacks, the intercistronic region between genes traM and traJ. In order to exclude the possible involvement of genes other than finP which are present on pSFI211, we synthesized gene ftnPand placed it in front of a lambda PL promoter. The construction is

Fig. 6. Expression of the artificial finP gene. Total RNA from E. coli cells containing the gene for a thermosensitive lambda repressor and pALi3 were extracted after growth at the permissive (30"C) or the nonpermissive f42''C) temperature. The RNA was electrophoreticalt-y separated, transferred onto a nitrocellulose filter and probed with a DNA fragment containing the finP gene.

RNA from R1 81

102%

100%

Calculated free energies of wild-type ond mutated stem-and-loop structure II (kcal/mol) -22.8

B

322

-168

-162

-202

65%

-21,5

presence of additional wild-type finP genes in the same cells. There appear to be three classes of mutants: those which do not deviate appreciably from the wild type, those showing an enhanced repressor activity, and mutants having lost it. The first category comprises one mutant in loop i, and two in stem II. The second category consists of mutants in stem II whereas mutants having partially or completely lost repressor activity are those with an altered loop II. In addition to mutants affecting flnP directly a construct was tested with wild-type finP but with a deletion removing both oriT and the traM promoters (Koronakis e( al.. 1985). This deletion mutant was tested because of the possible effects of traM transcripts upon finP RNA function (see the Discussion for details). Like the loop II mutants mentioned above, it shows enhanced repressor activity. The finP transcript contains two possible ORFs for short poJypeptldes. Two of the mutations interrupt the first of the two reading frames by introducing stop codons, i.e. mutants m4 and m7. The second reading frame is interrupted in mutants m5 and m6. As all four mutants show either an unchanged or an increased repressor activity the possibility that the ftnP product is a polypeptide can be definitely excluded. When the mutated ftnP genes were introduced into cells containing plasmid Rl, a very marked derepression by at least two orders of magnitude of the conjugation frequency was observed in two cases, i.e. in the two mutants with an altered loop II (m9 and miO). No other mutant f/nP genes influenced the conjugation frequency of Rl in a significant way (Table 2).

111

Fig. 7. A. The predicted secondary structure ot the finP RNA molecule. Single base ctianges in the produced finP mutants are indicated. Ttie boxed AUGs show starts ot potential finP ORFs. B. A histogram of ttie relative mating efficiencies ot R1-19 with ditterent co-resident plasmids is shown. Abbreviations of the coresident plasmids are 322 (pBR322); 111, p B R i n {w\ finP), 110,p8R110(o/vTand fraM promoters deleted); m4 to mlO, p B R I I I m4tom10(ftnPmulants).

Mutagenesis of finP and function of the altered moleoules Gene ftnPcarried by the 1.2kb Bgl\\/Pst\ (2) fragment was altered by site-specific mutagenesis in such a way that four single base changes were introduced in stem II, two single base changes in loop II and one base change in loop I of the predicted secondary structure of FinP (Fig. 7). The mutated fragments were cloned into pBR322 and transformed into ceils containing either R1-19 or R l . The conjugation frequencies were subsequently assayed and compared to a control with only the vector plasmid present. A histogram showing the results of quadruplicate measurements which are calculated as the percentages of the uninhibited control, is presented in Fig. 7. The frequency of R1-19 conjugation is reduced four-fold by the

Discussion The best documented examples for antisense RNAs acting as repressors in prokaryotes are RNA I of the C0IEI replication control system and RNA-OUT of the insertion sequence IS?O (Tomizawa, 1984; Kleckner, 1989). Here we demonstrate the involvement of another antisense RNA. namely the finP RNA, in the regulation of conjugal DNA transfer of plasmid R l .

RNA nature of i\nP Originally it was shown by Ostermann et ai. (1984) that a Table 1. Conjugation frequencies of pDB12 in the presence of pAL13. Incubation temperature (°C) growtfi

mating

30 30 35 35 42 42

30 35 35 42 35 42

per donor cell 0,28 2.62 7.68 4.58 0.18 0.17

82

G. Koraimann et al.

TaWe 2. Conjugation frequencies of Rl from £ co/i MC1061 to £. coli J5. Co-resident plasmid

Number ot transconjugants per donor cell

PBR322 pBRIIO pBRin pBRIII m4 pBRnim5 pBRIII me pBR111m7 pBR111m8 pBB111m9 pBRIIImiO

5.6 X l O - " 1.0 X 10 ^ 2.0 X 10 ' 2.0 X 10 ' 4.2 X 10 ^ 2.3 X 10 3 1.7 X 10 ^ 3.0 X 10 3 6.0 X 10 ' 1.3 X 1 0 '

DNA segment from R1 carrying the finP gene represses the conjugation o1 derepressed Rl derivatives in trans. Although several authors have suggested (Willetts and Maule, 1985; Dempsey, 1989; Paranchych ef al-, 1986; Frost et al., 1989) that the finP product of related IncFII plasmids acts as an RNA, this assumption still lacks convincing evidence. The RNA nature of FinP is clearly shown by the results obtained here, with site-specific mutants that interrupt potential open reading frames in the finP gene and still repress the conjugation system.

Repression of conjugation by the expression of a synthetic i\nP gene No genes, or parts of genes, around finP contribute to this activity because the expression of a synthetic finP gene alone suffices to repress conjugation. The repression obtained with pDBI 2 as a conjugative test plasmid is very marked and in contrast to experiments with natural finP expressed from its own promoter. pOB12 is a multicopy derivative of Rl -19 and therefore requires higher intraceilular concentrations of FinP for significant repression in trans, which only the artificial gene linked to the powerful lambda-Pu promoter can supply. The product of the synthetic gene finP is active in this case although the transcript is significantly longer than the natural one. There are five additional ribonucleotides at its 5'-end which are due to the construction of the recombinant plasmid and a 3'-segment of approximately 400 nt originating from vector sequences. The transcription terminators following the vector (3-lactamase gene (Balbas et al., 1986) are probably responsible for the formation of the two transcripts, measuring 400 and 570 nt. Hence, in this experimental context, the artificial 3' -terminus of the finP RNA is not relevant for finP RNA function. Although no experimental data are available which indicate that the second stem-and-loop structure of finP RNA functions as a rho-independent transcription terminator, it nevertheless strongly resembles such a structure (Platt and Bear. 1983).

Reasons why we could not detect a 72-nt transcript in the expression system might therefore be the incomplete nature of the termination signal of the synthetic gene, the use of a different promoter (Telesnitsky and Chamberlin. 1989), or the experimental procedure employed to detect the transcripts. At this point we would like to stress that the observation of the 72-nt finP RNA alone does not imply transcription termination after the predicted stem-andloop structure II of the finP RNA. There might exist a longer primary transcript which is then rapidly processed or degraded to give rise to the RNA molecule of the observed length. Finally, both mechanisms might be operational.

Interaction of FinP with its target mRNA and the functions of domains of FinP Although the experiments described do not directty investigate the mechanism of the interaction between FinP and FinO and its target (the 5'-end of the traJ mRNA including its RBS) the in vivo tests of finP mutants allow some important conclusions to be drawn. It is clear from the experiments with the mutants that nucleotide exchanges in loop II lead to loss of repressor activity. This correlates with reports on natural finP alleles where the changes are almost exclusively located in loop II (Paranchych e/a/.. 1986). An exchange ofa base in the middle of the loop eliminates repressor activity completely, while an exchange at the right border of the loop retains it at least partially. One mutation at the border of loop I behaves like a wild type. Thus the primary interaction with traJ mRNA seems to occur mainly through loop II, a proposal substantiated by the derepressing effect of these mutations on Rl conjugation. The experimental data from this study together with the fact that all natural frnP alleles differ in loop II strongly support a model in which the interaction between finP RNA and traJ mRNA occurs by a mechanism similar to that proposed in the 'kissing model' for the binding of RNAI to RNAII of the ColEI plasmid (Tomizawa, 1984). Other well characterized antisense RNA-target mRNA regulatory systems in which the effects of loop mutations have been investigated are CopA/CopT of plasmid R1, and RNA-OUT/RNA-IN of IS ?0 (Persson ef al., 1988; Kittle ef a/.. 1989). The recognition specificity of CopA RNA seems to reside solely in loopll(Perssonefa/., 1988); the four mutations in RNA-OUT that severely reduce pairing with wild-type RNA-IN map to the upper portion of the loop domain, whereas mutations in the stem domain of RNA-OUT do not have any effects on the pairing ability (Kittle et ai.. 1989). Mutations affecting stem-andloop I or stem II of FinP also do not seem to hinder antisense RNA-mRNA hybrid formation. Results obtained in this study with non-loop II mutants and a study of F plasmid finP mutants previously presented (Frost ef ai., 1989) support this assumption. When base exchanges

imp RNA from Rl were introduced into stem II either no significant change or an increase in repressor activity was observed relative to the wild-type gene. Mutations m4 and m7 are of the former type, and m5 and m6 of the latter type. No clear correlation could be found between the repressor activity of the mutants and the calculated AG values (shown in Fig. 7) of the altered stem-and-loop structures II. The three- to eight-fold enhancement of repressor activity of mutants m5 and m6 over the wild type is puzzling. Possibly the facilitated dissociation of the RNA helix of stem II in these mutants as a necessary prerequisite to the formation of the RNA-RNA hybrid with traJ mRNA leads to increased repressor activity.

83

activity in trans but do interact with FinO and, as a result of their high concentration, remove the limiting wild-type finO product from the system. As a consequence, wildtype finP RNA supplied from Rl is unable to form the complexes with FinO needed for efficient repression and is probably for this reason degraded at a higher rate (Frost et al., 1989). The wild-type FinP concentration within the cells drops and repression of conjugal transfer is relieved. Our interpretation is in accord with results obtained with studies in the F plasmid where the site of interaction between FinP and FinO was found to reside in stem I (Frost etal., 1989). Experimental procedures

Transcription from traM promoters interferes with FinP repressor activity

Bacterial strains, piasmids, phages and growth conditions

If the finP gene is supplied in trans on the recombinant plasmid p G K I I I the transfer rate of R1-19 is reduced four-fold. This inhibition is still greater in the case of plasmid pGK110 which contains the wild-type finP gene but lacks the or/f sequences and the two fraM promoters. If one assumes transcription starting from the two traM promoters and RNA polymerase partially reading through into traJ sequences this phenomenon can be explained. The 5'-segment of the traJ mRNA is supplied by transcnpts starting from at least three promoters, namely its own and from the two traM promoters. In the latter case partial read-through into traJ coding sequences has to be assumed. The additional transcripts formed from the recombinant plasmid pGK111 are able to sequester finP RNA and thus decrease the concentration of the free molecules available to act in trans upon the traJ mRNA of R1-19. In support of our interpretation are the data ot Dempsey (1989), who detected traM transcripts of plasmid R100 that reach into fraJ coding regions. Based on this finding he proposed that transcripts from the two traM promoters provide additional sense RNA to compete with traJ mRNA for finP RNA which would connect trad expression not only to the presence of FinP but also to transcription from the traM promoters.

The bacterial strains, plasmids and phages used in this work are shovi/n in Table 3. E. co//cells were grown in 2 x TY medium (16g tryptone, lOg yeast extract, 5g NaCI per litre). Antibiotics, when needed, were added at the foliowing final concentrations: epicillin, 100)tg ml ': chloramphenicol, 30hJLg ml '; tetracycline, 15p.gml '; kanamycin, 70(i.g ml '.

FinO and FinP When conjugation of R1-19 or its derivative pDB12 is tested we assume that repression occurs without the supportive effect of the finO gene product. Therefore, if the concentration of finP RNA is high enough, partial repressor activity occurs even without FinO. With R l . the repressed wild type, as conjugative plasmid in the assay, we found a drastic derepression by at least 100-fold when the loop II mutants were present on multicopy plasmids in the same cells. We interpret this result to mean that the mutant finP RNA molecules are unable to exert repressor

Enzymes, chemicals and oligonucieotides DNA polymerase I large fragment (Klenow fragment), Hybond M&G, Hybond N, T4 DNA polynucieotide kinase and all radioactively labelled compounds were purchased from Amersham International, Reslnction enzymes RNaseA, RNase T1 and T4 DNA ligase were purchased from Boehringer Mannheim GmbH. 'RNasin' RNase-inhiDitor and reverse transcriptase were from Promega Biotec. Oligonucleotides were synthesized on an Applied Biosystems 381A DNA synthesizer.

DNA manipulations Recombinant DNA techniques were performed according to Maniatis (1982), or following the manufacturers' protocols. Transformations were carried out using either the standard CaCl2 protocol or a shorter version (Golub, 1988),

Computational sequence analysis RNA sequence analysis including the prediction of the potential secondary structure of the finP RNA and the calculation of the free energies were done by the algorithm of Zuker and Stiegler (1981) which is included in version 6.1 of the GCG Sequence Analysis Software Package (Devereux etal.. 1984).

RNA preparations Metabolic labelling of RNA. E. co//J5(R1)or£". co//J5(R1-19) were grown in [^^P]-orthophosphate-containing medium, according to Garen and Levinthal (1960). Ten milliCuries were added per litre of medium. The growth procedure and the separation by gel electrophoresis was described earlier (Koraimann and Hogenauer, 1989). After identification by autoradiography the 4S RNA

84

G. Koraimann et al.

Table 3. Bacterial strains and plasmids. Strain/Phage/Piasmid

Relevant genotype/characteristics

Source/Reference

Sandoz Forschungsinstitut, Vienna, Austria N. Minton/Casabadan and Cohen (1980)

5K

lacP, ci587ts, lac Lambda", pro, met hsdR2, hsdM', hsdS ', araD139. Maraleu)7697, \(\ac)X74. galE15. galKW. rpsL. mcrA. mcrSI hsdR. hsdM'. tre. thi, lac, rpsL

Ml3 phage M13KO7

Km'', p15A, ori

J. VieiraA'ieira and Messing (1987)

Plasmids R1 R1-19 pDB12 pBR322 pACYC177 pUC119

IncFII, Ap", Cm", Km''. Sm", Su". fi* IncFII, Ap", Cm", Km", Sm'', Su", ff" IncFII, Cm" multicopy derivative of R1-19 Ap". Tc" Ap", Km" Ap",/ac'IP0Z',M13. ori

T. Leisinger, ETH Zurich, Switzerland Blohm(1979) J. Vietra/Balbas et al. (1986) Chang and Cohen (1978) J. VieiraA/ieira and Messing (1987) Ostermann etal. (1984)

Escherichia coli W3110 J5 MCI 061

pSFI210 pSF(211

Ap", Tc", 7.7kb EcoRI fragment E of R1 cloned into EcoRi of pBR325 Tc". Psfl-cut and recircularized pSFI210. This construct contains the 5.3kb EcoRI/Psfl (1) fragment shown in Fig.

W, Goebel, Universitat Wurzburg, FRG

Ostermannefa'. (1984)

i r.

PSFI216 PSFI218 pAL13

pGKIIO pGK111 pGKIII m4 through miO pBRIIO pBRIIl

pBRm m4 through miO

band was cut out and eiuted. The specific activity was between 2.5 and 3.5 x 10^c.p,m. ^ i g - ^ Preparation of non-labelled RNA. RNA preparations for Northern blotting, RNA-RNA-protection experiments and for primer extension experiments were performed by the standard hot-phenol method described by Miller (1972).

Southern hybridization CsCI-purified DNA from plasmid pSFt211 was digested with EcoRWPsti, from plasmids pSFI216 and pSFI218 with HincW and the fragments separated on 0.8% agarose gels. Two blots on nitrocellulose filters were prepared and probed with 10^ c.p.m, cm ^ of /n-v/Vo-labelled 4S RNA. One blot was probed with RNA

Tc", 2,6kb HincW (2)/£coRI fragment from pSFI211 cloned into pBR325 Tc", 1,3l(b HincW fragment cloned into pBR322 Km", synthetic lambda PL promoter and finP gene cloned into Bam HI/Ps(l-cut pACYC177 Ap", Bal31 deletion cloned in pUC119 (see text) Ap", 1.2 kb eg/ll/Ps(l(1} fragment cloned intopUC119 Ap", site-specific finP mutants of pGK111 Tc", Psfl/fcoRI fragment from pGKl 10 cloned into pBR322 Tc", Psd/fcoRI fragment from pGKl 11 cloned into pBR322 Tc". PstUEcoRl fragment from pGK111finPm4 through miO cloned into pBR322

Ostermann etal. (1984) Ostermann et al. (1984) This work

This work This work This work This work This work This work

from E. CO//J5(R1), and the other with RNA from E. co//J5(R1 -19). The hybridization was performed using standard protocols.

RNA-RNA protection experiment Two pSFI210-derived fragments, the 921 bp Xba\/Pst\ (2) and the 460bp EcoRV/Psfl (2) segments were cloned into the polylinker of plasmid pSP64. After re-isolation of the plasmid from E. coli cells the DNA was linearized with EcoRVorwith Psfl. The inserted DNA was transcribed in vitro as described by Melton et al. (1984) using (i-p^P]-GTP as radioactive label. The radioactive RNA was hybridized with total cellular RNA from f. coli 5K(pSFI210) by dissolving the nucleic acids in 80% formamide, 40mM PIPES, pH 6,7,0,4 M NaCI and 1 mM Na2-EDTA (ethylenecjiamine tetraacetic acid), and incubating the mixture first for 5 min at 80°C. then for 8h

finP RNA from R1 85 at 42°C. Subsequently, single-stranded RNA molecules were digested with 40 (xg ml ' RNase A and 2 |xg ml ' RNase T1 in the presence of lOmM Tris-HCI, pH 7.5, 5mM Na2-EDTA and 300mM NaCI for 1h at 30"C. After phenolization, the hybridprotected RNA was precipitated with ethano) in the presence of tRNA as carrier. The double-stranded RNA was dissociated by heating to 95"C and single strands separated by electrophoresis on a sequencing gel (6% acrylamide, 7M urea).

Primer extension Four hundred nanograms of the electrophoretically purified oligonucleotides 5'-AAAAATTGAAACTGAAAATC-3' and 5'GTCCATAGAATCCTTAACGG-3' were phosphorus 5'-endlabelled and desalted over a SephadexGI 00 column. They were mixed with 100 M-g of total RNA from E. coli cells carrying various plasmids. The mixture of RNA and oligonucleotide was dissolved in20p,l lOmM Tris-HCI, pH 8.0, 1 mM Na2-EDTA, heated for 10 min at 9O''C, then for 10 min at 43X. and finally mixed with 0.83 fil of 'RNasin', RNase-inhibitor (30U \).\ '), 5 (i-l 1 M Tris-HCI, pH 8.1, 5^1 0.1 M dithiothreitol and 7^.1 I M KCI. This solution was incubated for 2h at 43^C. Subsequently, 1 \x.\ 0.2M MgCb, 2.5^.1 each of a 20mM solution of all four deoxyribonucleotides and 40 units reverse transcriptase were added. After incubation of the mixture for 1 h at 43''C the RNA was hydrolysed by the addition of lOM-lof I M NaOH, 10^1 of 0.1 M Na2-EDTA, and 40 til of water and further incubated for 30 min at 70°C, The mixture was subsequently neutralized, phenolized and the reaction product precipitated with ethanol. Subsequently it was electrophoretically separated on an 8% polyacryiamide/7M urea gel.

Construction of pAL13 containing the synthetic finP gene The oligonucleotides required for the lambda PL promoter and the finP gene were purified using Applied Biosystems OPCs (Oligonucleotide Purification Cartridges). One of the four oligonucleotides (Oligo 3 in Fig. 5) was phosphorylated on its 5'-end so that after re-association of the complementary oligomers two doublestranded molecules were formed, only one earring a phosphomonoester group. The re-association reaction was done by slowly cooling down from 100°C an equimolar mixture of the complementary oligonucieotides, dissolved in ligation buffer. Subsequently 90 pmoles of the re-associated pL-promoter oligonucleotides and 90 pmoles of the ftnP oligonucleotides were mixed and ligated with 2 U of T4 DNA-ligase at 4" C overnight. The reaction product was purified by electrophoresis on a 0.8% low melting-point agarose gel, the 165bp band cut out, and the DNA isolated. This short DNA fragment was subsequently ligated with a 3020bp Pst\/BamH\ fragment ot ptasmid pACYC177. The resulting plasmid, which conferred kanamycin resistance to E. coli cells, was designated pAL13. The correctness of the sequence of the synthetic insert was verified by digesting pAL13 DNA with SamHI, labelling its ends either in the 5' position or in the 3' position with a-pP]-ATP or with a-pP]-dATP, digesting again with Psfl. isolating the small 165bp long fragment by electrophoresis on a Nu Sieve agarose gei and sequencing it after adsorption to Hybond M&G paper by the chemical procedure.

Expression of the finP gene from pAL 13 and analysis by Northern blotting pAL13 DNA was transformed into E. coli W3110 cl587ts and the celts were grown in 2 x TY medium at either 3O''C or 42°C. After centrifugation and resuspension of the cells in a sodium dodecyl sulphate-containing buffer they were broken by sonication and the RNA extracted as described above. The total RNA was heat-denatured and separated by electrophoresis on 3% Nu Sieve agarose gels in the presence ot formaldehyde. Ten and 20 M-g samples were applied. The RNA bands were blotted on a Hybond N nylon membrane, the membrane was treated with salmon-sperm DNA and Denhardt's solution and subsequently hybridized with the radioactive probe. The probe was prepared by cleaving pSFI210 DNA with B5/II and EcoRI, separating the fragments by agarose gel electrophoresis, isolating the 4,7 kbp fragment and synthesizing copies by means ot random priming in the presence of the Klenow fragment of DNA-polymerase I and t