¢;ene, 86 (1990) 35--43 Elsevier

35

GENE 03353

F a c t o r s affecting expression o f the r e e F gene of E s c h e r i c h i a c o i l K-12 (Recombinant DNA; recombination; DNA repair; post-translational control; phage ~.; overproduction; regulation)

Steven J. Sandier and Alvin J. Clark Department of Molecular Biology, University of California at Berkeley, Berkeley, CA 94720 (U.S.A.) Tel. (415)642-5827 Received by S.R. Kushner: 27 February 1989 Revised: 10 June 1989 Accepted: 30 July 1989

SUMMARY

This report describes four factors which affect expression of the reeF gene from strong upstream ~ promoters under temperature-sensitive clAt2-encoded repressor control. The first factor was the lone ~mRNA leader sequence consisting of the Escherichia coli dnaN gene and 95% of the dnoA gene and ~.bet, N (double amber) and 40~o of the exo gene. When most of this DNA was deleted, Reef became detectable in maxicells. The second factor was the vector, pBEU28, a runaway replication plasmid. When we substituted pUC118 for pBEU28, Reef became detectable in whole cells by the Coomassie blue staining technique. The third factor was the efficiency of initiation of translation. We used site-directed mutagenesis to change the mRNA leader, ribosome-binding site and the 3 bp before and after the translational start codon. Monitoring the effect of these mutational changes by translational fusion to lacZ, we discovered that the efficiency of initiation of translation was increased 30-fold. Only an estimated two- or threefold increase in accumulated levels of ReeF occurred, however. This led us to discover the fourth factor, namely sequences in the recF gene itself. These sequences reduce expression of the recF-lacZ fusion genes 100-fold. The sequences responsible for this decrease in expression occur in four regions in the N-terminai halfofrecF. Expression is reduced by some sequences at the transcriptional level and by others at the translational level.

INTRODUCTION

The reeF gene of E. coli K-12 maps at 83 min on the' E. coli chromosome in a group of genes which participate Correspondence to: Dr. AJ. Clark, Department of Molecular Biology, University of California at Berkeley, Berkeley, CA94702 (U.S.A.) Tel. (415)-642-0985; Fax. (415)-643-9290. Abbreviations: Ap, ampieillin; bp, base pair(s);/~Gai, p.gaiactosidase; dNTP, deoxyribonucleotide triphosphate; ELISA, enzyme-linked immunosorbent assay; kb, kilobase(s) or 1000 bp; Kin, kanamycin; NEM, N.ethylmaleirnide; nt, nueleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PAGE, polyaerylamide-gel electrnphoresis; PMSF, phenylmethylsulfonyifluoride; Pollk, Klenow (large) fragment of E. coil DNA polymerase I; R, resistance; RBS, ribosome-binding site; ReeF, reeF-encodedprotein; SDS, sodium dodecyl sulfate; UV, ultraviolet; wt, wild type; XGal, 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside. 0378-1! 191901503.50© 1990ElsevierScience PublishersB.V.(BiomedicalDivision)

in DNA metabolism. These genes are dnaA, which encodes a replisome assembly protein for initiation of chromosome replication at oriC (Bramhill and Kornberg, 1988), dnaN, which encodes the/~ subunit and processivity factor of DNA polymerase III (McHenry et al., 1986), reeF, and gyrB, which encodes the/3 subunit of DNA gyrase (Menzel and Gellert, 1987). These genes seem to be both coordinately and separately regulated (Armengod and Lambies, 1986; Menzel and Gellert, 1987; Wang and Kaguni, 1987; Armengod et al., 1988). Blanar et al. (1984) have hypothesized that dnaN and recF are translationally coupled. The recF143 allele (Horii and Clark, 1973) was originally detected by the conjugational recombination deficiency it conferred on a recB21 recC22 sbcBl5 sbcC201 strain (Lloyd and Buckman, 1985). In a recB + recC + sbcB + sbcC + background, although conjugational recombination is not significantly affected (Horii and Clark, 1973),plasmid

36 recombination is reduced (Kolodner et al., 1985; Cohen and Laban, 1983). This difference in phenotype appears best reconciled by the hypothesis that Reef is substratespecific in its operation and that the conjugational substrate can be processed along a recombinational pathway independent of RecF. Substrate specificity may also account for the differential effect of reef mutations on UV mutagenesis (Ciesla et al., 1987). Additional effects ofrecF mutations on cell survival after UV include repair of daughter-strand gaps and double-strand breaks (Wang and Smith, 1988) and derepression of about 3/4 of the lexA regulon genes tested (Thorns and Wackemagel, 1987). The reef143 also affects genes induced during the adaptive response (Volkert, 1989). Volkert et al. (1984) found that recA441 suppressed a recF mutation with regard to UV sensitivity. Volkert and Hartke (1984), Wang and Smith (1986), and Thoms and Wackemagel (1988)then found mutations in srfA, mapping close to rec,4, which partially suppressed UV sensitivity of reef mutations. One allele, s~'A 803, has now been def'mitively located in recA (Madiraju et al., 1988) and renamed recA 803. The in vitro properties of RecA803 suggest that RecF helps RecA deal effectively with secondary structure and Ssb bound to long-lived single-strand DNA substrates (Madiraju et al., 1988). Consistent with this, overproduction of Ssb protein confers a RecF- phenotype (Moreau 1987; 1988). Testing these hypotheses about reef function requires that the Reef be characterized. Blanar et al. (1984) determined the nt sequence of reef and identified its protein using the maxicell technique. This paper describes: (1)a series of plasmids in which the ,vt.vR promoters of phage have been used to express the Reef in maxicells; (2)attempts to overproduce Reef by modulating the sequences which affect initiation of translation; and (3) subsequent analysis of three additional factors which limit Reef production in vegetative cells.

MATERIALS AND METHODS All bacterial strains used in this work were derivatives of E. coli K-12. ABlI57 has the following partial genotype: thr-I leu-6 thi-1 lacY1 ara-14 xyl-5 mr/-1 proA2 argE3 ~sL31 tsx-33 and supE44. JCllg03 is ABll57 with reef143 (Horii and Clark, 1973). JC2941 is a ~ lysogen of ABll57. CSR603 is ABll57 thr + leu + fecal uvrA6 and phr-I (Sancar et al., 1979). Phage ~ninL4 NTN53 c/857 nin5 was given to us by William Dove (Inokuchi and Dove, 1978). ~clg0-was a gift from Harrison Echols. ,all restriction enzyme digestions and ligations were done with enzymes purchased from New England Biolabs using recommended conditions. All constructions were verified by restriction analysis. All strains containing plasmids were maintained at 30°C. UV tests for detection of reef + plasmids were done by quantitative survival curves (Horii and Clark, 1973). Transformation mixtures whose DNA had been subject to the site-directed mutagenesis protocol were used to transform JC2941. Site-directed mutagenesis was done according to Zoller (1984) as modified by Kunkel (1985). Oiigos used in this work are shown in Table I.

RESULTS AND DISCUSSION

(a) Expression of recF in maxicells Our major objectives when beginning this work were first to identi~ and then overproduce the Reef preparatory to its purification. Our strategy involved the following: (1) providing conditional expression in case overexpression of Reef might be lethal; (2)circumventing the possible occ,rrence of repressor control by amplifying the number of copies of the reef gene in the cell; (3)using a strong

TABLE I Mutagenic oligo primers" Name

Oliso primerb

Plasmid with mutation©

prSJSl prSJS3 prSJS$ prSJS6 prSJS9 prSJSl I

AATGAGACTCTTATOAGCCT CACC GACCACCATAAAOGGCCCGCTCT A TG T T G T C A T CT CGAGGAAACTCTTA AGT CGAGGAOGAAGAAATGAGCCT GOCGG C C C.QAATT.~.CAGAAOA TTATCTCCCOOGT TTAACTT

pSJSI22 pSJSI26 pSJS 130 pSJSl40 pSJS 142 pSlSl$9

" Oligoswere synthesizedby Bruce Malcolmof the UC BiochemistryDepartment and then purified by gel electrophoresis(20% polyacrylamidegel containing 8 M urea). The part of'the gel which contained the olige was then excised and the oligo eluted by overnightincubationin 0.5 ml of buffer containing0.5 M ammoniumacetate/10mMmagnesiumacetate.Thissolutionwasthenfilteredthrougha 0.45Izmfilterto removepiecesofpolyacrylamide and the filtratewas passedthrougha Css sep-pakcolumnpurchasedfromMillipore.The columnwas then washedwith I ml ofwater and the oligoeluted with I ml of 60% methanol.This columnfraction was then evaporatedto dryness and then resusponded in 20 pl of'water. b Bases underlinedare those not complementaryto the wt sequences. c Mutationswere introduced into the plasmid by the method described in Fig.4, legend.

37 (27479)1"1 A (27887)

tl.3

I[ • :int xis

(3286"7)

('349.52) .

(3689.5)

B H .

.

. A

[ ninL4 I.[~-.t/x_x_\xx_x-,.-,...-x\~.,l -I|"bla orlt tet pSJSI exo bet ~ • pBR322

•

%

int xis

.

'bto o.;

! t t

t

pSJS2

"~'m i • I % t •

'

cro,clAt2 rex ,| / / / / / / H H / / / / / / / / / / I ~ pRK248 clAt2 II ] u pRK248 Ostos)ai m.t ~ ~s'ao)

t

! t

" i,u xis

tu Y ..'°exo bet [ nial.4

t t

"'"

NTN53 I"

I

40% exo

"bet

I ]

n

uu

]32%cro Ic/At2

pSJS6

"'..aE [

I

nbd.4 ~=.P L (A2080 bp)

P RM ~

"~ ~

pSJS9 ~J ,a,r I ~

]'1

" "

"

26'% rexA

PR Fig. I. Construction of pSJS6. The numbers in parentheses show the position of the restriction enzyme site relative to 2. DNA as defined by Daniels et at. (1983). Restriction enzyme sites are: A, Aml; B, agill; E, EcoRi; H, Hindlll. Crosshatched boxes represent either pBR322 (Bolivar et al., 1977) or pRK248 sequences. The blackened boxes show the sequences fi'ompRK248e/ts which have replaced the DNA between the Bgili and Hindlil sites in pSJS2, pSJSl was constructed by digesting ~lnL4 NTN$3 c!857 nin5 D N A with HindlIl, isolating the 7.4-kb Hindlll fra~pnent and mixingit with Hindlll-digested pBR322 DNA as vector. In the clone chosen, the 7.4-kbfragment was oriented so that the Pt. promot(:r was closest to the £eoRl site of pBR322, pSJS1 was then digested partially with Aml to delete a l.i-kb fragment encoding part of the Int geno and part of the let gene and one Hlndlll site. This plasmid was called pSJS2. Finally,pSJS6 was constructed by replacing a small B&lll.tllndlil fragmentofpSJS2 with a larger Bglil.ilindlll fragmentof pRK248 eli8 (Bernard and Helinski, 1979). The replacement contained clAt2, a temperature sensitive allele of the ~1 8ene and promoter Pn. (N.B, The cl 8ene found on pSJS6 contains a Hindlll site not found in the wt cl gene. This site is located at approximately the same position as the llindlll site created by the Ind-mutation. This new site may have been created by the mutation ciAt2.) in this case the transformation mixture was spread on LB mediumagar (Miller, 1972)containing I mM MgSO4, 50/AgAp/ml and prespread with 109pfu of 2.el90. Incubation at 300C permitted selection of transformants inheriting 2. immunity. Besides elAt2 pSJS6 contains the Pt. and Pa promoters oriented to initiate transcription in the same direction and a doubly mutant alleleof the N gene (suppressed in a supE44 strain).

promoter to drive transcription, and (4) enlisting an antiterminator to neutralize the influence of possible transcription terminators. To implement this strategy we used a temperature-dependent runaway replication plasmid pBEU56 as vector containing a fragment encoding recF, dnaN and 95 % of dnad (Uhlin et al., 1983), We then inserted an EcoRl fragment from pSJS6 (Fig. 1) containing the ,~ regulatory genes clAt2, OL PL, Ca Pa and N (double amber) making pSJS9 (Fig. 2). In maxicells, this plasmid produced six proteins of 59, 47, 42, 27, 25 and 23 k D a at 40°C (Blanar

E

( f

/gs~ -

A

lilt B

] I ~ . [ l ~ l j IdAel

Maxicells Wimle cells

-

-

'

PL PR ..-..--_..

-

-

-

-

pSJS40

+

-

pSJS42

+

-

nd

+

PRM L

pSJS20 ! L

~

1 pSJSl20 (pUC118)

i 'bla ori .^ I x~,\...,.....,.,l [ annl II at ""-pBR322

..°.°°°

E .."

S IIIISPPA

1

pSJSI3 III

exo bet ] ninL4 i--=~\\\\\\\\.l "oc It, pBR322

pemein

ReeF 40-kDa

E

I

Fig. 2. pSJS9 and the construction of its deletion derivatives. The restriction enzymesites on pSJS9 are: A, Avail; B, Bglll; E, EcoRI; H, Hkal; P,/~ull; S, Sau3Al. The blackened boxes represent the extent of sequence deleted from pSJS9. All plasmids shown except pSJSI20 used pBEU28 as their vector, nd, not determined, reeF was placed under the control of c/At2 by digesting pSJS6 DNA with EcoRI, isolating a 3.3-kb EcoRl fragmentcontainingthe c/At2, 2.Pt. J,npromoters,NTN$3,bet,and part of exo, mixing it with partially EcoRI digested DNA of pBEU$6 (Uhlin et al., 1983),treating the mixture with DNA ligase, transforming JC11803 (Horii and Clark, 1973) and selecting at 30°C for 2.immunity and Kma. Clones were tested for the orientation ofthe inserted fragment by restriction analysis.The plasmidchosen, pSJS9,contained the ~L Pa promoters oriented so as to transcribe dnaN and reeF.pSJSI3 was made by deleting an EcoR! fragmentcontaining reef frompSJS9, pS/S20 was made by deleting a Pmll fragment from dnal¢. Other derivatives were made from pSJS20 DNA by partial digestion with restriction enzymes and isolation of appropriate DNA fragments. These frzxgnents were treated with DNA ligase and used to transform JC! 1803.Transformers were selected on LB agar plates containing !mM MgSO4,$0/AgKm/ml and spread with 109 ~c!90. All plasmids shown except pSJSI3 restored full UV resistance to a recFI43 strain at 30°C. et al., 1984). A deletion analysis ofpSJS9 showed that these proteins were the gene products of the exo-dnaA fusion gene, a pBEU28 encoded gene, dnaN, bet, aphA and c/At2 genes, respectively (data not shown). No protein was found which correlated with the recF gene. Because of the similarity in the expected mass of the DnaN (42 kDa) and R e e f (40 kDa), the 42-kDa band could have contained either or both proteins. To determine which, we deleted a 591-bp Pvull fragment from pSJS9 to make pSJS20 (Fig. 2). This deletion removed only dnaN DNA, eliminated the 42-kDa band and produced the predicted 22-kDa band resulting from fusing N- and C-terminal coding sequences (data not shown). No new band which could have been the R e e f was seen (data not shown). We were particularly surprised not to see a RecF band because sequences identified by Armengod and Lambies (1986) as inhibiting RecF expression from the recF promoter were removed by the P m I l deletion. Thus we concluded that additional sequences blocked detectable R e e f production in maxicells. To remove these putative sequences, we made plasmids pSJS42 and pSJS40 by partial digestion of pSJS20 D N A with Hhal and then ligation (Fig. 2). Both plasmids encoded a 40-kDa protein identified by Blanar et al. (1984) as the product of recF.

38

Comparisons of the fusion points of the deletions upstream from reeF in pSJS40 and 42 revealed that both deletions maintained an ORF whose N-terminal portion was encoded by the N gene and whose C-terminal region was encoded by the dnaN gene. Thus, the two plasmids which produce the 40-kDa RecF in maxicells also maintain the overlapping stop and start codons to allow translational coupling between the mutated dnaN and the wt recF genes. Co) Expression o f recF in whole ceils

Having detected expression of recF in maxicells with pSJS42, we tested its expression in whole cells by looking

I

,, la ,r----It i 30 40 30 40 30 4 0 30 4 0 30 4 0 °C

kl)o

at accumulated protein by Coomassie blue stain after 3 h at 40°C. Fig. 3 shows that no 40-kDa protein accumulated. Thus we concluded that to detect RecF in whole cells we had to identify other factors which inhibited overproduction. To begin this identification we changed vectors, since the pBEU28 vector in pSJS42 (which kills cells by runaway replication at 37°C or higher) forced us to grow cells at 30°C (Uhlin et al., 1983). We excised pBEU56 from pSJS42 and replaced it with pUC118, producing plasmids pSJS 120. Without expecting there to be any difference in RecF accumulation in strains carrying pSJS42 or pSJS 120, we compared extracts obtained 3 h after turning on transcription at 40°C (Fig. 3). Detectable quantities of RecF were present in the strain with pSJSI20 at 40°C. The reason for this is not known. One possibility is that some gene product on pBEU28 not contained on pUC118 prevented accumulation of RecF at 40°C. If there is some such product, it does not prevent function of RecF at 30°C. We found that pSJS42 restored full UV resistance to a recF143 strain at 30°C indicating that the recF gene was expressed (data not shown). By contrast pSJSI20 only partially restores UV resistance at 30°C and does no better at 40°C where RecF was detectable. (c) Increasing the efficiency of initiation of translation of the recF gem

43--

"

/

,~

+++++,+++ ++:+ +

+,

.~.-RecF

25-I

2

3

4

5

6 7 8 g 10

Fig. 3. Visualization of accumulation of RecF protein in whole cells by Coomassie blue stain. The figure shows the results of 0.1% SDS-12% PAGE ofprotein extracts which have been normalized for equal numbers of ceils. Cells were incubated at 30°C in L medium until they were in midlog phase. ! ml ofcell culture was removed, centrifuged, and the pellet resuspended in 0.1 ml final sample buffer (60mM Tris pH6.8/2% SDS/0.25 M ,#-mercaptoethanol/20 v/v glycerol) plus protease inhibitors: I mM NEM/I mM PMSF/I mM EDTA (Sigma Chemical Company)/0.01 mM pepstatin/l ~8 per ml aprotinin (Boehringer Mannheim Biochemicals). The remainder of the culture was shifted to 40°C and incubated for 3 h. The cells were diluted periodically to maintain logphase growth. Of this culture I mi was treated as above to measure the accumulation of RecF. Extracts were electrophoresed according to the method of Laemmli and Favre (1973). The molecular size markers were: ovalbumin (43 kDa) and carbonic anhydrase (25 kDa).

It is known that several factors can affect the efficiency of initiation of translation. These are the length and structure of mRNA leader sequence (Gold, 1988), the RBS (Gold, 1988) and the 3 bp before (Hui et al., 1984) and alter (Looman et al., 1987) the AT(} start codon. From the nt sequence, we determined that the recF construction in pSJS 120 had an mRNA leader sequence of 362 nt with several potential start codom upstream from the reef start codon, a poor match to the consensus RBS sequence and nonoptimal sequences before and after the ATG. We wanted to optimize the efficiency of initiation of translation by sequentially changing the reeF translation initiation signals by site-directed mutagenesis. After each change, the translational initiation signals of recF and the first 50 bp of recF were translationally fused to the seventh codon of lacZ. These constructs were then tested for their ability to produce #Gal.

The wt recF construction contained on pSJS 120 (lacZ derivative pSJS 133) gave a rate of #Gal production of 3.8 units/min/A (Fig. 4). Changing the codons before and after the ATG to a more optimal configuration (Hui et al., 1984; Looman et al., 1987), respectively, had little effect on/~Gal production [pSJS 122 (lacZ derivative pSJS 134)]. We then changed the RBS to be more homologous with the consensus RBS (Steitz, 1979). This too had little effect on the rate of /~Gal production [pSJS130 (lacZ derivative

39 Name of plasmid recF

recF-lacZ

Sequence of parts of recFI and recF

pSJSl20 pSJS133 CAT

Rqm

Itmc~p~

&8~: 0.7

GCC bAT GAG ACT GTA ATG TCC

ATG 2.'/:1:0.1

pSJS122 pSJSI34 CAT GCC APT GAG ACT CTT ATG AGC

0~

01

i

RBS $.8:1:0.6

pSJSI30 pSJS136 CAT CTC GAG GAP, ACT CTT ATG AGC

1

mRNA leader

pSJS132 pSJS137 AAA.GTC GAG GAA ACT CTT ATG AGC

i tlt llt pSJSI40 pSJS144 APA GTC

GAG GAG GPA GAA ATG AGC

ATG and RIBS

42 :l:&.q 120±26

Fig. 4. Evolution ofscquences from pSJS ! 20 and pSJS 140 by site-directed mutaganesis. This figure shows the mutations made in the translationinitiation region (reeF/, reef leader) of the reef gene contained on pSJSI20. Short downward arrows indicate mutations, pSJSI20 was constructed by cleaving pSJS42 and pUCi 18 (Vieira and Messing, 1987) with £coRl, mixing the DNAs and treating with DNA ligase. Apn clones were screened for orientation of the insert in pUC118. One was picked so that the dAt2 gene was closest to the Hindlll site of the polylinker region. This plasmid was called pSJSI20 and was used as the starting material for site directed mutaganesis. To facilitate screening of the clones, each mutaganic oligo was designed so that a new restriction site was created, pSJSI32 was created in a two step process from pSJSI2I Using prSJS3, an Apal site was placed so that the first G in the Apal recognition sequence was at 35654 in the A sequence as defined by Daniels et al. (1983). To make pSJS126, in a subsequent step, prSJSS added an Xhol site adjacent to the RBS to make pSJSI30. This plasmid was then restricted withXhol andApal, treated with Pollk and then with DNA ligase to create pSJS132. The long downward arrow marks the junction point created by the deletion of the Xhol-Apal fragment. To monitor how the mutations which we introduced into our constructions affected the efficiency of translation, we fused each changed translational initiation region to lacZ. This was done by restricting each plasmid with gstll and EcoRI (in some cases gmal which also cuts in the polylinkar ofpUC118) and treating the DNA with Pollk in the presence of dNTPs. This DNA was then mixed with gmal.digested pFRI09 (Shapira et al., 1983), treated with DNA ligase and used to transform ABII57. Transformants were selected for Ap a using the ~ immunity selection. These clones were then screened for their ability to produce white colonies at 30°C and dark blue colonies at 40 ° C on minimal media containing XOal purchased from Sigma Chemical Company as well as having the expected restriction enzyme pattern. Thus pSJS 120,126, 130, 132, 140 were used to make pSJSI33, 134, 136, 137, 144, respectively. ,6Oal assays were done as described in Miller (1972) using the SDSchloroform procedure for rendering the cells permeable. Cultures were incubated at 30°C until the cells reached midlng phase when duplicate zero time samples were taken. The cultures were then shifted to 40°C. Two samples were taken at 30, 60 and 90 min. The results of duplicate samples were averaged and slopes calculated. Final results represent the averages of at least two experiments and typically more than four. Standard deviations were typically less than 10% of the average.

pSJS136)]. The next step shortened the mRNA leader sequence from 362 to 44 bp removing all upstream potential start codons before the reef start codon. We found that this additional modification increased the rate of/~Gal production to 42 units/min/A [pSJS132 (lacZ derivatives pSJSI37)]. We then made one more modification to the sequence between the RBS and the ATG. We changed this

sequence to a purine-rich sequence. We saw a substantial increase in the rate of~Gal production to 120 units/mln/A [pSJS140 (lacZ derivative pSJS144)] (Fig. 4). Thus we were able to increase the efficiency of initiation of translation of the recF gene by 30-fold. However, we found that pSJS140, which contained the 30-fold translationaHy optimized start codon gave only about twofold higher amounts of ReeF (Fig. 3) than did pSJSl20. (d) Sequences inside the reef gene inhibit its own expression Because the amount of Reef in extracts of pSJS 140 cells did not seem to exceed that in pSJS 120 cells by the amount expected from the 30-fold difference in rates of A~Gal formation, we decided to test the possibility that sequences within reef inhibited its expression. To locate such sequences we made a set of plasmids in which increasing lengths of the reef coding sequence were fused to lacZ. All of these plasmids have the translation initiation region of pSJSI40 and are diagrammed in Fig. 5A. The rate of/~Gal production was 120 tmits/min/A with pSJSI44 which contained only 50 bp of reef fused to lacZ. This contrasts with a rate of 1.3 units/min/A which was obtained with pSJSI54 which contained 543 bp of reef fused to lacZ. This experiment indicates that the nt sequences contained in the intervening 500 bp of reef DNA lower the rate of /~Gal production and may also lower expression ofwt reef. To determine which sequences were involved, we tested three other translational fusions with endpoints at bp 72, 142, and 390 of reef. The more reef DNA present in each of the fusion genes the lower the rate of/IGal production. To be sure that there were no inhibitory sequences in the second half of reeF, a fusion at 1028bp was made (pSJS 156). This fusion led to 3.2 units/min/A of pGal. The slight increase in expression from 1.3 units/min/A (pSJSI54) could be due to either the two in-frame ATGs in the second half of the gene which could be used as start codons or factors which enhance reeF expression.

(e) Does protein instability play a role in the inhibition of recF overexpression ? The preceding experiment did not distinguish whether any combination of transcription, translation, mRNA or protein stability was affected by reef sequences. To test protein stability we determined the half-lives of RecF-pGal fusion polypeptides from pSJS 144, pSJS 158 and pSJS 154 (data not shown). The half-life for the RecF-pGal polypeptides encoded by these three plasmids was approx. 15-30 min. Thus we concluded that instability of RecF/~Gal peptides was not responsible for the decrease in rate of pGal production we observed from the fusion plasmids.

40 A. recF.lacZderivativesof pSJS140(optimizedRBS) TramcdptionalFusions Translational Fusions [cl^t~ [p,_-I recFII I Plasmids ~of~ecF RatcofpGd DNA

IxSJSi44 pS~162

50 "72

1204-26 7S :t: 8

~ ~

psJslss

1~2

9~ ± I

~

i~ISlY2

390

S.4 :t: 0.1

[ ~

pSLS154

543

!.3 • O.S

pS~150

1028

3.2 ± 1

pSJS]65 ixsJSr;2 "Z---J ~ ~

Prod.

50 72

375:1:25 38S ± 40

m

~st~s

t42

uo ±

pSJSi73

390

149 ± I

ixSJS167

543

~PJ :1:2

pSJS174

1028

183±10

pSJS184 ixSJSl8.5

50 543

B. recF-lacZderivativesof pSJS40(wt RBS) / ixS~116 pSlS54

50 543

11.1 ± O.S 2.S ± O.S

~ ~

431 ± 43 90 ± 10

Fig. 5. RatesofllGal production as a function ofrecF DNA. (A) Plasmids originally derived from pSJSl40. (B)Plasmids originally derived from pSJS40. The constructions are as Follows.(1) recF-lacZ translation fusion plasmids. A series of six plasmids was constructed to fuse different extents ofrecF to the seventhcodon oflacZ. All ofthese plasmidscarried the optimized translation initiation region of' pSJSl40. One of' these, pSJS144,derived directly from pSJS140,and $0 bp ofrecF fusedto lacZ as describedabove. Another plasmid,pSJSI$2, had 390 bp ofreef fused to lacZ and was derived in three steps. First, an EcoRI cleavage site was introduced into pSJS 132 (Fig. 4) by sequence-specific mutagenesis using the ofigo(prSJSg). Second, DNA of.this plasmid, pSJS 142, was digested with BamHl + Noel. The larger of'twofragments representing the vector less the rAt2 PL Pa regulatory region and the translation initiation region of reef was purified and added to the purified small BamHI.N¢oi fragment derived from pSJS 140. DNA ligase treatment, transformation, selection and screening resulted in a clone carrying pSJSlS0. The presence of'theoptimized translation initiation region of'pSJS 140 was verified by restriction nucleese digestion. Finally, pSJSIS0 was digested with £¢oR1 to obtain a 2.3-kb fragmentcontaining ¢lAt2, PLPn, the optimized translation initiation region of'pSJS140 and 390 bp oFreef. This fragment was cloned using £coRl.cut pF'RI09 DNA as vector to make pSJSI52. (2) pSJSI52 was used as a vector plasmid for fragment exchange with three fusion plasmids Formec~zYompSJS40, in this exchange the fusion region ofpSJS 152 DNA was deleted by SJdl digestion and the equivalent fusion regions from Ssdl-dlaested pSJS 128, pSJS$4 and pSJS ! 18 DNAs (see below)were substituted, These plasmids had, respectively, 1028, 543 and 142 bp of'reef fused to lacZ. Since an $~dl cleavage site exists in lacZ and at bp SOin reef, each substitution event converts pSJS 152 into a plasmid with a difYerent reef lacZ fusion. These new plasmids were called pSJS156, pSJSI$4 and pSJSl$8, respectively. Their nature was verified by restriction nuclease digestion of DNA from Apa~.Imm÷ transformants of' AB1157. (3)Two of' the intermediate plasmids for the above constructions, pSJS128 and pSJSI$4, were prepared by cloning fragments of pSJS40 DNA using pFRl09 as vector. To make pSJS 128 a 3.0-kb£coRI.F.vpll frngmentofpSJS40 DNA was purified, treated with Pollk in the presence of dNTPs and then added to pFRI09 plasmids DNA digested with Sinai. To make pSJS$4, pSJS40 was partially digested with Sau3Al. Fragments in the runge of' 2-3 kb were purified and added to DNA of pFRI09 (Shapira et el., 1983) digested with BamHl. In both cases DNA mixtures were treated with DNA liaase and used to transform ABiI$7 to ApK and Imm+A. In each case plasmid DNA from AB I 1~7 transf'ormants was screened by restriction nuclease analysis to detect plasmids with the appropriate inserts. Restriction mapping showed that the £¢oRI-Bg/ll fragment containing the rAt2 8erie of' pSJS54 was identical to pSJS40. Hence the other $ou3Al site must be contained in pBEU28 sequences. (4)The third intermediate

(f) Do mmscriptioml factors inhibit recF overexpressiem? Is it possible that some recF sequences may cause transcription termination or m R N A instability? To test these possib'dities, we constructed a cognate set ofplasmids similar to the translational fusion plasmids, except that lacZ was incorporated with its own translational initiation region. With such plasmids, the rate of/~Oal production would indicate how ollen RIqA polymerase successfully completes transcription of the recF sequences and how stable the m R N A was, rather than how efficiently the translation of the reeF sequences occurred. The transcriptional fusion, p S J S 165 containing the first 50 bp ofrecF D N A , gave a level o f 375 units/,/600/rain (Fig. SA). The next fusion, p S J S I 7 2 , with the first 72 bp of recF D N A gave approximately the same rate (Fig. 5A). Therefore the

plasmid, pSJS118, was derived from pSJS54 and pFR97, pSJS54 DNA was digested with Ncol+ X~I. A 2-kb fragmentwas purified and treated with Pollk in the presence of' dNTPs. H~dlll-diBested DNA of'pFR97 (Shapira et al,, 1983) was similarly treated, the two DNA preparations were mixed, treated with DNA figase and used to transform AB1157 to Apa 2,Imm ÷. DNA minipreps from the transformants were screened by restriction endonuclease analysis to obtain a plasmid with the insert oriented appropriately to create the reef lacZ fusion pne. (5) The sixth fusion plasmid, pSJSI62, was made in three steps. DNA from pSJSl40 was subjected to site.specific mutapnesis using prSJSl I. The resulting plasmld, pSJS159, contained a CG--, GC transversion at nt 72 in reef. By this change a Smal cleavap site was created between codons 23 and 24 of reef and codon 24 wae changed from GGC to OGO, both ofwhich encode glycine. An Ncol-Bg/ll bngmant, containing this new Smal site and the c/At2, PL PR Banes plus the optimized translation initiation region from pSJS140, was purified and substituted For the cngnate Ncol-8~ll fragment of'pSJS$4 using standard procedures. DNA of'the resulting plasmid pSJSI61 was digested with Smal to delete 471 bp of reef and make the appropriate re~.lacZ Fusion. Restriction nuclease analysis was used to veri~ the nature of the final plasmid pSJS162. (6) pSJS40 contains the reef wt RBS. Several reef.lacZ translational fusion plasmids were constructed using pSJS40 as the progenitor. These were pSJS54, 116, 118, and 128. Constructions for all but pSJSll6 are given above, pSJSll6 was constru~ed by restricting pSJS$4 with Sstll + Sinai, treating with Polik in the presence of dNTPs and then incubating with DNA ligase. This DNA mixture was used to transform AB1157 and Ap~ colonies were selected. Clones were tested for their abilityto produce white colonies at 30°C and dark blue colonies at 40°C on plates containing XGal. One plasmid was saved, pSJSlI6, which fused the first 16 codons oFre~ to the seventh codon oflacZ. This fusion point was identical to those in pSJS133,134,136,137 and 144. (7) Transcriptional Fusions of' Reef to lacZ. A cngnate set of transcriptional fusions were constructed from the set of.translational fusions by restrictins the translational fusion plasmid with £¢oRI, isolating the £¢oRI fi'aament containing the r a t 2 gene and reef sequences and mixingthat with pRS415 (Simons etal., 1987)restricted with £¢oRI. The DNAs were treated with DNA ligase and used to transform AB1157. Colonies were selected as described For pSJS6 and the orientation determined by restriction endonuclease mapping. In this way pSJS54,116,144,162,158, 152, 154, and 156 were used to make pSJSI8$, 184, 165, 172, 166, 173, 167 and 174,respectively.,0Galwas assayed as described in Fig. 4 legend.

41 l.$-fold decrease in expression seen in the translational fusions with the equivalent amounts of m:F DNA is not due to transcriptional phenomena. In a similar fashion we observed that pSJ S 166, a transcriptional fusion containing the first 142 bp of reef DNA yielded a rate of 130 units of ~Oal/A 600/rain (Fig. 5A), which was about threefold less than the transcriptional fusion with the first 72 bp of recF DNA. The cognate translational fusions-showed an eightfold decrease in gene expression. Therefore, of the total decrease contributed by this region of DNA, about threefold was due to transcriptional phenomena. Similarly, most of the remaining eigh~old decrease in gene expression seen with the translational fusions of sequences between 142 bp and 543 bp of recF DNA cannot be attributed to such phenomena because the rate of 0Oal production decreases only l.$-fold in the transcriptional fusions

(Fig. 5A). (g) r e ~ - I ~ Z translational and transcriptional fusions using wt vecF From the work presented above, we wondered if the differential expression we observed when presenting the reef gene with optimum translation initiation signals was due to some attribute ofmcF which normally acts to regulate its expression or some new effect(s) not normally utilized. If' the effect is new, this would only be seen because of the optimum translation initiation signals we introduced. To decide between these two hypotheses, we compared the ability of vecF-lacZ fusions with wt translational initiation signals to produce #Oal (Fig. SB). We found that there was a fourfold difference in expression between translational fusions with 50 and $43 bp ofrecF DNA (pSJS 116 and $4). The fourfold decrease in expression between the translational fusions was mirrored by an equal decrease in ~Oal production in the transcriptional fusions (pSJSI84 and 185). Therefore, the decrease in expression seen in the translational fusions with the wt initiation of translational signals can be accounted for by an increase in termination oftrunscription or mRNA instability. Hence it appears that the optimum translation initiation signals cause a new effect not normally used to regulate ~cF expression.

(h) Conclusions In this study we have found the following: (1)Cloning recF downstream from a strong promoter using a runaway replication plasmid as vector was insufficient to overproduce RecF either in maxicells or in whole cells; to obtain expression of vecF in maxicells, we needed to eliminate most ofthe leader sequence consisting of parts of two genes from E. coli and parts of three genes from phage Z; we did not test whether the sequences inhibitory to recF expression

were in £. co//or ,I DNA. (2)The plasmid which led to RecF production in mexicells did not do so detectably in whole cells. Undetectable production in this case was due to the vector pBEU28. C h a n M the vector to pUC118 led to detectable RecF. (3)The initiationof ~ translation was not optimal, W e overcame thisfactorby changing the m R N A leader,the R B S and the tripletsin the vicinityofthe A T G translationalstartcodon. This, however, improved RecF production very littlebecause several sequences witl~n the vecF gale decreased expression. W e dissected the firsthall"of mcF into rq~ons and measured theireffectson both transcriptionand translation oflacZ. Region I Cop 50-72) when added to the first 49 bp of reef decreased translation 1.6-fold but had no effect on transcription. Region 2 Cop 73-142) when added decreased transcription roughly threefold and probably contributed proportionally to the eightfold decrease in translation. Adding region 3 Cop 143-390) essentially did not affect transcription but lowered translation roughly twofold. Finally, region 4 (bp 391-543) reduced transcription about l.$-fold and probably contributed proportionally to the fourfold decrease in translation. Thus all four regions decrease translation and two affected transcription. Armengod and Lambies (1986) have previously suggested that there were sequences in dnaN and in reef which inhibit reef expression at the transcriptional level. In one part of this work, we eliminated the sequence in dnaN which they had hypothesized was inhibitory but we saw no stimulatory effect in maxicells. We also tested the region in vecF (bp 284-354) identified as a sequence-inhibiting transcription ofgaIK from two reef promoters (Armenlp~ and Lambies, 1986) and found no significant chanp in transcriptional expression of lacZ (Fig. 5). The difference between our results and those of Armengod and Lambies (1986) may be due to the differences either in promoters or reporter pnes. We compared the ability of recF-lacZ translational and transcriptional fusions to produce #Oal as a function of the translational initiation sequences and the amount of reef DNA. We found that in both cases (optimized vs. wt), the amount of expression from translation or transcriptional fusions with 50 bp of reef DNA was greater than that of fusions with 543 bp of reef DNA. In the nonoptimal case, the decreases in the transcriptional and translational fusions were equal and in the optimized case they differed by 25-fold. Optimization of the translation initiation signals did not affect the termination of transcription and/or mRNA stability throughout the first half' of the gene. The question then arises, what is limiting reef overproduction when the translational initiation signals have been optimized? As Blanar etal. (1984) pointed out, ~,¢F contains a high frequency (43.3 ~ ) of nonoptimal codons so that a limiting amount of charged tRNAs for nonoptimum

42 codons may inhibit overexpression. Spanjaard and Van Duin (1988) showed that two adjacent nonoptimum arginine codons will cause the ribosomes to shift reading frames 50% ofthe time, Although this situation occurs in region 4 of r e e f [at codons 132 (AGA) and 133 (AGA)] and can explain the decrease in expression due to translation in that region, it does not explain the decreases seen in other regions. It is possible that other combinations of adjacent nonoptimum codons could cause the ribosome to shift reading frames in the other regions as well. Such a hypothesis is consistent with the observation that increasing the rate of initiation of translation fails to increase the overall amount of expression. There may be a physiological explanation for why there are many built-in sequences to limit r e e f expression by both at the level of transcription and translation. One possibility is that R e e f is highly toxic so that cells have several mechanisms to avoid producing any more than is absolutely necessary. Preliminary data on the viability of cells containing p S J S l 2 0 and pSJSl40 contradict this hypothesis. Further experiments to establish this await quantitative measurements of ReeF by ELISA or enzymatic assay. A second possibility is that R e e f is highly specialized for particular functions and that control factors (perhaps positive control factors) are required to elevate reeF expression. Inhibition ofgene expression by sequences within a gene as measured by translational fusion to lacZ has also been reported for the uncB gene (Solomon etal., 1989) and recQ gene of £, cog K-12 by Irino et al, (1986), Since the recQ and reef genes both participate in the R e e f pathway of recombination (lrino et al., 1986), the two mechanisms of inhibition of gene expression encoded by regions of D N A within these genes may be similar and may be found in other R e e f pathway genes,

ACKNOWLEDGEMENTS We are grateful to Nelle Neighbor-Alonzo for typing the research was supported by grant NP-237 from the American Cancer Society and PHS grant AI-05371 from the National Institutes of Health.

manuscript. The

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