THE JOURNAL OF BKX.OGKXL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 21, Issue of July 25, pp. 12300-12305,199O Printed in CJ S. A.

Effects of Transcription and Translation on Gyrase-mediated DNA Cleavage in Escherichia coli*

(Received for publication, January 4, 1990)

Hyeon-Sook Koo$, Hai-Young Wu, and Leroy F. Liu§ From the Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Gyrase-mediated DNA cleavage on plasmid DNAs was measured in Escherichia coli treated with oxolinic acid. On pBR322 DNA, gyrase cleavage sites were concentrated in the region between the 3’-ends of the tetA ahd bla genes. The preferential cleavage in this region was dependent on RNA transcription and the divergent orientation of these two transcription units. The enhanced gyrase cleavage also required transla- tion; chloramphenicol treatment or the insertion of a translation terminator within the 5’-proximal region of the tetA gene abolished the enhanced cleavage. We suggest that the enhanced gyrase cleavage may reflect the changes in local DNA supercoiling during RNA transcription as gyrase cleavage in vitro was shown to be sensitive to the supercoiling state of DNA. The effects of transcription and translation on gyrase cleavage can best be explained by the twin-super- coiled-domain model of transcription (Liu, L. F., and Wang, J. C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7024-7027).

DNA supercoiling has been recognized to be an important determinant for many DNA functions including DNA repli- cation, RNA transcription, and recombination (l-4). How- ever, the mechanisms by which DNA supercoiling is generated and regulated in uiuo are not completely understood. Previous studies in prolraryotes have demonstrated the importance of DNA topoisomerase I (w protein) and II (DNA gyrase) in the maintenance of the supercoiling state of intracellular DNA. DNA topoisomerase I preferentially removes negative super- coils while topoisomerase II specifically reduces the linking number of a closed circular DNA (relaxation of positive supercoils or introduction of negative supercoils) (5, 6).

In addition to topoisomerases, RNA transcription has also been implicated as an effector of the supercoiling state of intracellular DNA based on a number of recent observations. First, highly positively supercoiled pBR322 DNA was pro- duced in Escherichia coli treated with gyrase inhibitors (7). Second, transcription of tetA greatly increased negative su- percoiling of pBR322 DNA in topA (w protein gene) deletion mutants (8). Third, studies in eukaryotic cells have also suggested that RNA transcription may require a topoisomer- ase activity (9-16). To explain these findings, a twin-super- coiled domain model has been proposed which describes the

* This work was supported by National Institutes of Health Grant GM27731 (to L. F. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a postdoctoral fellowship from Jane Coffin Childs Memorial Fund for Medical Research.

§ Recipient of the ACS faculty research award. To whom corre- spondence should be addressed.

mechanics of RNA transcription (17). In this model, translo- cation of an elongating RNA polymerase complex along right- handed double-helical DNA results in a relative rotation of the RNA polymerase complex around the DNA helical axis. The resistance to the rotational motion by the large RNA polymerase complex, due to the size and mass of the nascent RNA (including the associated proteins such as ribosomes) or the anchorage of the RNA polymerase-nascent RNA complex to a large macromolecular structure, leads to the rotation of the DNA helical axis. The rotation of the DNA helical axis produces positive supercoils ahead of and negative supercoils behind the RNA polymerase complex. This model has gained some support from recent studies on the formation of posi- tively supercoiled plasmid DNAs in E. coli (18), yeast (19), and an in uitro transcription system (20).

One strong prediction of the transcription model is the existence of variations in superhelical tension along a DNA molecule due to translocation of the elongating RNA polym- erase complex. To probe the local variations in DNA super- coiling during RNA transcription, we have analyzed the effi- ciency of oxolinic acid-induced, gyrase-mediated DNA cleav- age, which was shown to be sensitive to DNA supercoiling in vitro, for various plasmid DNAs in E. coli. Our studies showed that the efficiency of gyrase-mediated DNA cleavage was strongly influenced by RNA transcription and translation. In particular, enhanced DNA cleavage was observed in the region between the 3’-ends of the two divergently transcribed genes.

EXPERIMENTAL PROCEDURES

Plasmids and E. coli Strains-E. coli AS19 was originally isolated as a permeable mutant by selecting for its sensitivity to actinomycin D and was shown to be highly permeable to a number of other antibiotics including novobiocin (21). The construction of plasmids pBR322dT, pBR322dA, pBR322TI1, pBR322TI2, and pJW270 has been described (18, 22). pBR322APtetA and pBR322A (EcoRI-PstI) in the previous work (18) were renamed here as pBR322dT and pBR322dA, respectively, for convenience. Plasmids, pEV-X, pBa-X, pSa-X, pNr-X, which contain an inserted translation termination codon at EcoRV, BamHI, SalI, and NruI sites on pBR322, respec- tively, were obtained from Dr. Douglas E. Berg (23).

Enzymes and Chemicals-E. coli gyrase subunits A and B were obtained from Dr. Martin Gellert (National Institutes of Health). Restriction enzymes were from Bethesda Research Laboratories. Oxolinic acid, rifampicin, chloramphenicol, chloroquine diphosphate, and novobiocin were purchased from Sigma.

Isolation of DNA from Oxolinic Acid-treated E. coli Cells-Cells were cultured in either LB or M9 medium supplemented with casa- mino acids. Except for E. coli harboring pBR322dA which were grown in media containing tetracyline (12.5 pg/ml), all cells were cultured in media containing ampicilin (25 fig/ml) overnight. The overnight cultures were reinoculated (1 to 1000 dilution) into fresh medium without antibiotics and grown to 0.2 optical density at 600 nm. Oxolinic acid (10 pg/ml) was then added for 1 min during incubation. Where necessary, rifampicin (50 pg/ml) or chloramphenicol (200 pg/ ml) was added to inhibit RNA transcription or translation respec-

12300

RNA Transcription and DNA Supercoiling 12301

tively, 6 min prior to the addition of oxolinic acid. IPTG’ (1 mM) was added 30 min prior to oxolinic acid treatment where indicated. Oxolinic acid-treated cells were rapidly cooled in ice water, and then pelleted at 4 “C by centrifugation. Cells (25 ml culture) were resus- pended in 200 ~1 of an ice-cold solution (50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0), and lysed with 300 ~1 of 2.5% SDS. Alternatively, oxolinic acid-treated cells were lysed directly by adding an SDS-EDTA solution to the cell-culture according to the method of Lockshon and Morris (24), followed by DNA isolation as described. In both cases, 100 ng of a control plasmid DNA (about 15 kilobases in size) was added to the lysates to monitor the recovery of cellular plasmid DNAs in the following isolation process. In the case of pelleted cells, proteinase K (200 pg/ml) was added to the lysate, and the reaction was incubated at 37 “C overnight. After two phenol extractions and one chloroform extraction, RNase A digest.ion (100 pg/ml) was done at 37 “C for 20 min, followed by phenol extraction and ethanol precipitation. The DNA pellet was resuspended in 75 ~1 of TE (10 mM Tris, pH 8.0,O.l mM EDTA) and the DNA (10 ~1) was digested with restriction enzymes, followed by phenol extraction and ethanol precipitation.

Isolation ofl’lasmid DNAs from Nouobiocin-treated Cells-Plasmid DNAs were isolated from E. co/i AS19 treated with the gyrase inhib- itor novobiocin (100 pg/ml) using the alkaline procedure, as previ- ously described (18). To inhibit protein synthesis, chloramphenicol (200 pg/ml) was added 5 min prior to the addition of novobiocin.

Gel Electrophoresis-Gel electrophoresis was performed in 1% aga- rose gel with % x TPE electrophoresis buffer except for the gels in Figs. 1 and 6 where i/r x TBE buffer was used (25). Two-dimensional gel electrophoresis was performed as described by Wu et al. (18), with chloroquine diphosphate (12 pM) in the second dimension.

Mapping of C~rase Ueauage Sites by Indirect End Labeling-To map gyrase cleavage sites on plasmid DNAs, an indirect end labeling procedure was followed (26). The smaller fragment from EcoRI/SalI or AuaI/PuuII double-digestion of pBR322 DNA was nick-translated with [,r-‘LrP]dCTP and used as a probe in in situ Southern hybridi- zations (18). DNA samples were digested with the appropriate restric- tion enzymes (see figure legends) before electrophoresis.

In Vitro Gyrase-mediated Cleauage on Positiuel>j and Negatiu& Supercoiled Plasmid DNAs-Positively supercoiled pBR322 DNA was isolated from AS19 cells t.reated with novobiocin (100 pg/ml, 30 min) (18). Gyrase was reconstituted by mixing gyrase subunits A (3 pg, 24,000 units) and B (16 pg, 8,000 units) in 250 ~1 of the storage buffer (50 mM Tris, 0.2 M KCl, 1 mM EDTA, 5 mM dithiothreitol, 50% glycerol, 0.5 mg/ml bovine serum albumin). Supercoiled plasmid DNAs (600 ng in an 8.~1 reaction) were incubated in a reaction buffer (36 mM Tris, pH 7.6, 6 mM MgCl?, 5 mM spermidine, 5 mM dithio- threitol, 50 pg/ml bovine serum albumin, 100 pg/ml yeast core RNA, 1 mM ATP) in the presence of oxolinic acid (100 pg/ml) at 37 “C for 5 min. In the competition experiments, approximately equal amounts (300 ng each) of negatively and positively supercoiled DNAs were mixed together. A seriallv diluted E. co/i gyrase enzyme (2 ~1) was added to the DNA solutions incubated with oxolinic acid and the reaction continued at 37 “C for 30 min before being terminated with 1 ~1 of 10% SDS and 1 ~1 of 0.5 M EDTA. Proteinase K (2.0 pg/ reaction) digestion was done at 37 “C for 3 h.

RESULTS

E. coli DNA Gyrase Preferentially Cleaves Positively Super- coiled DNA in Vitro-Oxolinic acid has been shown to trap gyrase on DNA producing protein-linked double-stranded DNA breaks upon the addition of SDS (27, 28). To test whether oxolinic acid-induced gyrase cleavage of DNA is sensitive to DNA conformation, purified E. coli gyrase was reacted with negatively and positively supercoiled plasmid DNAs. When positively supercoiled pJW270 DNA and nega- tively supercoiled pBR322 DNA were combined and reacted with E. coli DNA gyrase in the presence of oxolinic acid, positively supercoiled pJW270 DNA was linearized more ef- ficiently (about 4-fold) (Fig. 1, lanes a-e). The preferential linearization of positively supercoiled pJW270 DNA was not due to its dissimilar DNA sequence, as positively supercoiled

’ The abbreviations used are: IPTG, isopropyl thiogalactoside; SDS, sodium dodecyl sulfate.

abcdefghij -. --. 4

pJW270 (III) - e *

pBR322 (Ill)-

pJW270 ii’, >

pBR322 (-) = e (+I

Flc,. 1. Effect of DNA conformation on oxolinic acid-in- duced, gyrase-mediated DNA cleavage in vitro. The samples in lanes a-e contained approximately equal amounts of negatively su- percoiled pBR322(-) and positively supercoiled pdW270(+) DNAs. The samples in lanes f-1 contained approximately equal amounts of pBR322(+) and pJW270(-) DNAs. Linear DNA is marked as (Ill). The gyrase concentration in each reaction is as follows: lanes e and], undiluted reconstituted gyrase; lanes d and i, 2-fold diluted ,yrase; lanes c and h, 4-fold diluted gyrase; lanes b and d, 8-fold diluted gyrase; lanes a and f, 16-fold diluted gyrase.

pBR322 DNA was preferentially linearized by gyrase in a reaction containing positively supercoiled pBR322 DNA and negatively supercoiled pJW270 DNA (Fig. 1, lanes f-j). These results suggest that the efficiency of gyrase cleavage induced by oxolinic acid is sensitive to the DNA supercoiling state and therefore can be used as an indicator of the supercoiling state of DNA in vivo.

The Efficiency of in Vivo Gyrase Cleavage Is Altered by Transcription-In order to study the effect of RNA transcrip- tion on the DNA supercoiling state, we examined oxolinic acid-induced gyrase cleavage of plasmid DNAs in vivo. The extent of oxolinic acid-induced (10 pg/ml, 1 min) linearization of pBR322 DNA was measured in E. coli AS19, a strain highly permeable to a number of antibiotics. As shown in Fig. 3 (compare lanes a and b), gyrase cleavage of pBR322 DNA as measured by plasmid linearization was reduced 2-fold in cells treated with the transcription inhibitor rifampicin (50 pg/ml, added 5 min prior to oxolinic acid), suggesting that RNA transcription enhanced gyrase-mediated cleavage of pBR322 DNA. To further assess the effect of active transcription on gyrase-mediated cleavage, the efficiency of cleavage was meas- ured on pJW270 DNA, which is a pBR322 derivative with the tetA promoter replaced by the IPTG-inducible lacUV5 pro- moter (see Fig. 2 for a schematic diagram). Upon the activa- tion of the tetA transcription by IPTG, linearization of the plasmid DNA was increased about 3-fold (Fig. 3, compare lanes e and g). Therefore, transcription appears to be neces- sary for the enhanced cleavage of pBR322 DNA by gyrase.

Divergent Transcription of the tetA and bla Genes Is Re- sponsible for the Enhanced Gyrase Cleavage-A number of pBR322 DNA derivatives (see Fig. 2 for schematic diagrams) were used to determine the individual effects of tetA and bla gene transcription as well as the effect of their relative ori- entation on gyrase-mediated cleavage in vivo. Inactivation of either tetA transcription (see plasmid pBR322dT which con- tains a small deletion in the tetA promoter) or bla transcrip- tion (see pBR322dA which has a -700 base pair deletion of the early part of the bla gene including its promoter and the 5’-flanking region) abolished the effect of transcription on gyrase cleavage (Table I). Furthermore, inversion of the tetA

12302 RNA Transcription and DNA Supercoiling

PI”,,

pBR322

PVUll

pBR322dT

tetA Q ori mm

*“aI PVUll

pBR322dA

pBR322Tll pBR322Tl2 pJW270

FIG. 2. Schematic diagrams of transcription units in the plasmid DNAs. Plasmid pBR322dT has a deletion in the tetA promoter and pBR322dA has lost part of the bla gene including its promoter by the deletion of EcoRI-PstI region in pBR322 (18). pBR322TIl and pBR322TI2 were made by inverting EcoRI-AuaI and EcoRI-PuuII fragments, respectively, from pBR322 (18). pJW270 was derived from pBR322 by replacing the tetA promoter with lac UV5 promoter, and by inserting the lacl gene which is transcribed from the I” promoter (22). The bla gene can also be transcribed from the antitet promoter which is not shown in the diagram (29).

pBR322 pBR322dT pJW270

rlrl I I

IPTG - - + + rU. -+ -+ rU. -+ -+

ab cd ef gh

Ill--- ‘-

I--p FIG. 3. Effect of transcription on gyrase-mediated lineari-

zation of plasmid DNAs. Plasmid DNAs were isolated from E. coli AS19 cells treated with oxolinic acid. Rifampicin was added 5 min prior to the addition of oxolinic acid to inhibit transcription, IPTG treatment was for 30 min before the addition of oxolinic acid to activate transcription of the inducible tetA gene on pJW270. I, ZZ, and III denote supercoiled, nicked, and linear forms of plasmid DNA, respectively. DI denotes the dimeric form of supercoiled plasmid DNA.

TABLE I The effect of RNA transcription on gyrase-mediated cleavage of

plasmid DNA in vivo % cleaved is the amount of linearized molecules relative to total

plasmid DNA. rif, rifampicin.

c/o cleaved Plasmid Cleavage ratio

rift-) rif(+) rift-)/rif(+)

pBR322 22 10 2.2 pBR322dT 4 LO 0.4 pBR322dA 12 17 0.7 pBR322TII 11 14 0.8 ~BR322T12 14 23 0.6

gene (see Table I, pBR322TIl and pBR322TI2) also abolished the enhanced cleavage observed with pBR322. These results are most consistent with the interpretation that simultaneous divergent transcription of the tetA and bla genes is essential for enhanced cleavage by gyrase. The rom gene, which also forms a divergent pair with the bla gene in pBR322dT and pBR322TI1, did not seem to affect gyrase-mediated cleavage, possibly due to the short size of the transcription unit. It is noted that except for pBR322 DNA and pJW270, RNA tran- scription in general reduced cleavage of plasmid DNAs by gyrase (see the cleavage ratios listed in Table I and compare lanes c and d in Fig. 3). Studies in E. coli DM800, a topA deletion mutant strain, using the same set of plasmid DNAs showed similar trends (data not shown). However, the effect of transcription on gyrase-mediated cleavage of pBR322 DNA is less pronounced in DM800 than in AS19 cells (data not shown).

The Enhanced Gyrase Cleavage in pBR322 DNA Occurs Primarily in the Region between 3’-Ends of the tetA and bla Transcription Units-An indirect end-labeling procedure was used to determine the sites of gyrase-mediated cleavage on plasmid DNAs (Fig. 4) (26). Cleavage sites on pBR322 and pBR322dT DNAs were mapped both clockwise from the EcoRI site (Fig. 4, panel A) and counterclockwise from the PuuII site (Fig. 4, panel B). Cleavage sites on pJW270 DNA were also mapped clockwise from the Hind111 site (Fig. 4, panel C). For pBR322 DNA, there was an overall increase in cleavage by gyrase during transcription. However, the major increase in cleavage occurred in the region between the two 3’-ends of the tetA and bla genes (nucleotide 1200-3200) (see Fig. 4, panels A and B). This polarized distribution of cleavage sites on pBR322 DNA was also observed by Lockshon and Morris (24). Similarly, for plasmid pJW270, activation of the tetA gene by IPTG induction increased overall cleavage by gyrase with the major increase appearing again in the region between the 3’-ends of the tetA and bla transcription units (Fig. 4, panel C). To quantitate the increased level of cleavage in different regions of the DNA, pBR322 DNA was arbitrarily divided into I3 regions and the regional cleavage ratios (-rif/ +rif) were determined based on the integrated intensities of bands in each region obtained by densitometric scanning of the autoradiograms (Fig. 5). The cleavage ratio showed a marked increase in the region between the 3’-ends of the two transcription units, as shown in Fig. 5A. For plasmid pBR322dT, RNA transcription reduced rather than increased overall cleavage by gyrase and no strong variation of regional cleavage ratio (-rif/+rif) was observed (Fig. 4, panels A and B, and Fig. 5B).

The Enhanced Gyrase Cleavage Is Not due to Changes in Overall DNA Supercoiling-The high regional cleavage ratios between the 3’-ends of the tetA and bla genes may reflect higher positive supercoiling (or lower negative supercoiling) in these regions of pBR322 DNA during RNA transcription. However, it is also possible that the overall supercoiling of PBR322 DNA may change during RNA transcription and the high regional cleavage ratios may have resulted from hyper- sensitivity of the cleavage sites in these regions to the change in the overall supercoiling. The latter possibility was ruled out by the following observations. First, the linking number of pBR322 DNA during transcription was not significantly different from that without transcription in AS19 cells; rifam- picin treatment (5 min) reduced the average linking number of the plasmid DNA only slightly (about three linking num- bers) (data not shown). Second, the gyrase cleavage sites in these regions were not particularly sensitive to the overall supercoiling state of the plasmid DNA, as shown in in vitro

RNA Transcription and DNA Supercoiling 12303

pBR322 pBR322dT

rlri or. - + - l

ab cd

2.4-

C IPTG - - + + rif. _ + _ +

abed

pBR322 pBR322dT

nrl rl?. - + - +

ab cd

FIG. 4. Mapping of in uiuo gyrase cleavage sites on plasmid DNAs. DNA samples were isolated from E. coli AS19 cells treated with oxolinic acid as described in Fig. 3. Panel A and B, lanes a and b: pBR322 DNA. Lanes c and d, pBR322dT DNA. Samples in lanes b and d were isolated from cells treated with rifampicin prior to oxolinic acid. Pane/ A, DNA samples were digested with EcoRI and probed with nick-translated EcoRI-Sal1 fragment of pBR322 DNA. Panel R, DNA samples were digested with PouII and probed with nick-translated P&I-AuaI fragment. Panel C, pJW270 DNA. Acti- vation of tetA transcription on plasmid pJW270 was achieved by IPTG addition. Plasmid DNA was digested with HindIII, and gyrase cleavage sites were mapped using nick-translated EcoRI-Sal1 frag- ment of pBR322 DNA. Lane a, control. Lane b, rifampicin only added. Lane c, IPTG only. Lane d, IPTG and rifampicin. The numbers at the left side of the gels indicate the positions (kilobase) on the pBR322 map.

gyrase cleavage studies using negatively and positively super- coiled pBR322 DNAs (Fig. 6, lanes c and d). In general, the cleavage patterns of the negatively and positively supercoiled DNAs were very similar; only minor changes were observed (marked by solid circles and triangles).

The Effect of Translation on Gyrase-mediated Cleavage- According to the twin-supercoiled-domain model, coupled transcription/translation is important to provide sufficient viscous drag to cause rotation of the DNA helical axis and hence the generation of supercoiling waves (17). To test the effect of translation on gyrase-mediated cleavage of plasmid DNAs, two different sets of experiments were performed. First, the protein synthesis inhibitor chloramphenicol (200 rg/ml, added 5 min prior to oxolinic acid) was shown to have the same effect as the transcription inhibitor rifampicin (Fig. 7, compare lanes b and c) in reducing gyrase-mediated cleavage of pBR322 DNA. Second, the gyrase cleavage ratio (-rif/+rif)

&lA ori pBR322

bla

B ggl 22 ;E ;A g8 $@ 0 1000 2000 3000 4000 bp

or, pBH322dT

FIG. 5. The regional gyrase cleavage ratios on plasmid DNAs in cells treated without and with rifampicin. The pBR322 (panel A) and pBR322dT (panel B) DNAs were arbitrarily divided into 13 sections, and the overall cleavage efficiency within each region was determined by integrating the intensities of all the bands measured by densitometric scanning of the autoradiograms in Fig. 4. The regional cleavage ratio refers to the ratio of cleavage efficiency on that region of DNA in cells treated without and with rifampicin.

was shown to parallel the length of the translated region of the tetA mRNA using a set of mutant pBR322 plasmids (23) with a translation terminator inserted at various positions within the tetA gene (Table II). These results suggest that coupled transcription/translation is responsible for enhanced cleavage of pBR322 DNA by gyrase.

The Effect of Translation on the Formation of Positively SupercoiledpBR322 DNA in E. coli Treated with Novobiocin- The formation of highly positively supercoiled pBR322 DNA in novobiocin-treated E. coli AS19 cells has been shown to depend on RNA transcription and the divergent orientation of the transcription units (18). The parallism between the positive supercoiling assay and the gyrase cleavage assay in the dependence on RNA transcription prompted us to test whether protein translation was also required for the forma- tion of positively supercoiled pBR322 DNA. As shown in Fig. 8, chloramphenicol treatment (200 pg/ml, added 5 min prior to novobiocin) significantly reduced the population of posi- tively supercoiled pBR322 DNA.

DISCUSSION

The effect of transcription and translation on the gyrase- mediated cleavage of plasmid DNA can best be explained by the twin-supercoiled-domain model of RNA transcription (17). As predicted by the model, coupled transcription/trans- lation in bacteria causes a large resistance to the rotational motion of the RNA polymerase complex (including its nascent RNA chain bound by ribosomes) and hence increased rotation of the DNA helical axis, which in turn produces positive and negative supercoils. However, fusion of positive and negative supercoils within the same plasmid DNA molecule is expected to occur rapidly unless the rotational diffusion pathways for

12304 RNA Transcription and DNA Supercoiling

in vivo in vitro nn a b c d

TABLE II

The effect of coupled transcription/translation on gyrase-mediated cleavage of olasmid DNAs in viva

* $li

t 1.2- r*==w-

3 2 LO-muy

FIG. 6. Mapping of gyrase cleavage sites in vitro and in vivo. Lanes a and b, pBR322 DNA samples were isolated from E. coli AS19 treated with oxolinic acid, or rifampicin and oxolinic acid, respectively. Lanes c and d, negatively (lane c) and positively (lane d) supercoiled pBR322 DNAs were separately reacted with purified E. coli DNA gyrase and oxolinic acid. Gyrase-cleaved pBR322 DNAs were purified and digested with EcoRI and hybridized with nick- translated EcoRI-Sal1 fragment of pBR322 DNA. A), indicates sites which are more efficiently cleaved on negatively supercoiled DNA. 0). indicates sites which are cleaved more efficiently on positively supercoiled DNA. The conditions for in vitro cleavage were as de- scribed under “Experimental Procedures” and undiluted gyrase stock solution was used here. It was noted that the extent of plasmid DNA linearization was the same for both positively and negatively super- coiled DNA when they were reacted separately with gyrase.

abc

ori

2 ‘-*

1

---o,

PI 1-o- * ’ -4

FIG. 7. Effect of protein synthesis on in vivo gyrase-me- diated cleavage. pBR322 DNA was isolated from E. coli AS19 treated with oxolinic acid. Lane a, control. Lane b, chloramphenicol added 5 min prior to oxolinic acid. Lane c, rifampicin added 5 min prior to oxolinic acid. DNA samples were digested with EcoRI and probed with EcoRI-Sal1 fragment.

these supercoils are somehow blocked. One possible mecha- nism by which the rotational diffusion of supercoils can be prevented is to anchor a DNA segment to a large macromo-

Plasmid Cleavage ratio rift-)/rif(+)

pEV-X 13 19 0.7 pBa-X 15 16 1.0 pSa-X 17 12 1.5 pNr-X 18 10 1.8

2nd D b

A B

n b. br i ;i; r

1 cr

a -9 a FIG. 8. Effect of protein synthesis on the formation of pos-

itively supercoiled plasmid DNA in E. coli AS19 treated with the gyrase inhibitor novobiocin. Plasmid DNAs were isolated using a published procedure and analyzed by two-dimensional gel electrophoresis (18). Panel A, chloramphenicol added 5 min prior to novobiocin (15 min). Panel B, no chloramphenicol; novobiocin (15 min). o-c, indicate highly negatively supercoiled, nicked, and highly positively supercoiled DNAs, respectively. The arc between a and c contains topoisomers of linking numbers in between those of a and C.

lecular complex. In the case of a plasmid DNA containing two divergently transcribed genes, each transcription unit during active transcription can act as an anchor for the other tran- scription unit. Furthermore, the supercoils generated by RNA transcription from each transcription unit enhance rather than cancel each other as is the case for parallel transcription units. Our present results are most consistent with the pos- sibility that the tetA gene and the bkz gene form a pair of divergent transcription units, and simultaneous transcrip- tion/translation of them leads to the formation of two oppo- sitely supercoiled domains on pBR322 DNA. The enhanced gyrase-mediated cleavage of pBR322 DNA in the region be- tween the 3’-ends of the tetA and blu genes may reflect the “positive” domain formed during divergent transcription of the two genes. The word positive is used in a relative sense to distinguish the two supercoiled domains. DNA in the positive domain may in fact be negatively supercoiled but less so than the other domain.

Our results on gyrase-mediated DNA cleavage parallel the studies by Wu et ul. (18). They showed that formation of positively supercoiled plasmid DNAs in E. coli AS19 cells treated with the gyrase inhibitor novobiocin was dependent on transcription. In both cases, divergent transcription of the tetA and bla genes has a major effect on these processes. In their studies, inactivation of either the tetA or the bla gene transcription, or inversion of the tetA transcription unit on pBR322 still produced positively supercoiled plasmid DNAs albeit at a much lower level. In our present studies, the cleavage ratios, rif(-)/rif(+), which vary from 0.4 (pBR322dT) to 2.2 (pBR322 DNA), can be taken as a quan- titative indicator of the effect of transcription on gyrase cleavage. The cleavage ratio is larger than 1 for such plasmids as pBR322 and pJW270 (with IPTG induction) with divergent tetA and bla transcription units (Table I and Fig. 3). The cleavage ratio is less than 1 for plasmids containing only one of the tetA and blu genes or both genes transcribed in the same orientation. The distinct effect of divergent transcrip-

RNA Transcription and DNA Supercoiling

tion on the cleavage ratio is also reflected in the marked enhancement of gyrase cleavage in the region between the 3’- ends of the tetA and blu genes on pBR322 DNA during transcription. These results support the notion that during divergent RNA transcription, two oppositely supercoiled do- mains are formed.

The negative effect of RNA transcription on gyrase-me- diated cleavage for the plasmids with transcription units in the same orientation cannot be explained. However, the uni- form decrease in cleavage on most gyrase sites in these plas- mid DNAs (as an example, see pBR322dT in Fig. 5) during RNA transcription may imply a global rather than local effect of RNA transcription on the plasmid DNAs, therefore, ruling out the possibility that the transcription elongation complex physically excludes gyrase from binding to sites only within the transcribed regions. The negative effect of RNA transcrip- tion on gyrase-mediated cleavage could be mediated by global change in DNA supercoiling. However, the linking number of plasmid DNA did not change significantly upon rifampicin treatment; a very slight decrease in plasmid linking number was observed within 5 min of rifampicin treatment for all plasmids tested.2 The lack of an explanation for the negative effect of transcription on gyrase cleavage could indicate the involvement of additional factors (e.g. binding of HU protein).

The effect of coupled transcription/translation and diver- gent transcription on gyrase-mediated cleavage is very similar to that on the formation of positively supercoiled plasmid DNAs in gyrase-inhibited cells, suggesting that the same basic mechanism may be responsible for both effects. However, the formation of highly negatively supercoiled pBR322 DNA in E. coli DM800 (AtopA) appears to require the transcription of the tetA gene but not the bla gene (8, 23). For example, pBR322dA which contains only the tetA transcription unit can be isolated from E. coli DM800 as highly negatively supercoiled DNA (18). However, no enhanced gyrase cleavage during transcription could be detected for the plasmid in the same bacterial strain.’ These results suggest that the forma- tion of highly negatively supercoiled pBR322 DNA in E. coli DM800, although similarly dependent on coupled transcrip- tion/translation, may require an additional as yet unidentified factor(s) or process(es). Lodge et al. (23) have recently shown that translation as well as transcription of the first 98 codons of the tetA gene is necessary for the formation of highly negatively supercoiled pBR322 DNA. It was proposed that the polypeptide translated from these codons anchors to the cell membrane and the rotation of RNA polymerase complex is restricted by the anchorage of RNA polymerase complex to the cell membrane through the nascent tetA mRNA and its cotranslational product (23). Whether membrane anchorage is an important determinant for the formation of negatively supercoiled pBR322 DNA in E. coli DM800 remains to be further investigated.

The supercoiling state of intracellular DNA is likely to be affected by many parameters besides topoisomerases and RNA transcription. At least part of the negative supercoiling in bacterial DNA has been shown to be constrained by his- tone-like proteins such as HU (30-32). RNA transcription appears to interfere with the binding of HU protein to DNA, as shown in the in vitro replication studies of X phage DNA and oriC-containing plasmids (33, 34). RNA transcription may affect the binding of histone-like proteins to DNA di- rectly, or indirectly through its effect on DNA supercoiling. The interplay among RNA transcription, topoisomerase ac-

’ H.-S. Koo, H.-Y. Wu, and L. F. Liu, unpublished results.

tion, and histone-like protein binding is likely to be crucial for the control of both global and local DNA supercoiling. The recent demonstration of supercoiling alteration during the movement of SV40 T antigen helicase may indicate ad- ditional complexities involved in supercoiling regulation (35). Studies of the effect of RNA transcription on DNA super- coiling may begin to unveil the complex mechanisms involved in supercoiling regulation.

Acfinowfedgments-We are very grateful to Drs. Jennifer Lodge and Douglas Berg for providing some of the plasmids, and Dr. Martin Gellert for E. coli GyrA and GyrB. We would like to thank Dr. Anette Bodley, Dr. Peter D’Arpa, Kawai Lau, and Hui Zhang for helpful comments on this manuscript.

REFERENCES

1. Cozzarelli, N. R. (1980) Science 207,953-960 2. Gellert, M. (1981) Annu. Reu. Biochem. 50,879-910 3. Liu, L. F. (1983) CRC Reu. Biochem. 15, l-24 4. Wang. J. C. (1985) Annu. Reu. Biochem. 54.665-697 5. Wang; J. C. (1971) J. Mol. Biol. 55, 523-533 6. Gellert. M.. Mizuuchi. K.. O’Dea. M. H.. and Nash. H. A. (1976)

Proc.‘Nak Acad. Sk. c. S. A. ?S, 3872-3876 ’ 7. Lockshon, D., and Morris, D. R. (1983) Nucleic Acids Res. 11,

2999-3017 8. Pruss, G. J., and Drlica, K. (1986) Proc. Natl. Acad. Sci. U. S. A.

83,8952-8956 9. Fleischmann, G., Pflugfelder, G., Steiner, E. K., Javaherian, K.,

Howard, G. C., Wang, J. C., and Elgin, S. C. R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,6958-6962

10. Gilmour, D. S., Pflugfelder, G., Wang, J. C., and Lis, J. T. (1986) Cell 44,401-407

11. Brill, S. J., DiNardo, S., Voelkel-Meiman, K., and Sternglanz, R. (1987) Nature 326,414-416

12. Egyhizi, E., and Durban, E. (1987) Mol. Cell. Biol. 7,4308-4316 13. Garg, L. C., DiAngelo, S., and Jacob, S. T. (1987) Proc. Natl.

Acad. Sci. U. S. A. 84.3185-3188 14. Stewart, A. F., and Schi& G. (1987) Cell 50,1109-1117 15. BriH, S. J., and Sternglanz, R. (1988) Cell 54, 403-411 16. Zhang, H., Wang, J. C., and Liu, L. F. (1988) Prod. Natl. Acad.

Sci. U. S. A. 85, 1060-1064 17. Liu, L. F., and Wang, J. C. (1987) Proc. Natl. Acad. Sci. U. S. A.

84.7024-7027 18. Wu, H. Y., Shyy, S. H., Wang, J. C., and Liu, L. F. (1988) Cell

53,433-440 19. Giaever, G. N., and Wang, J. C. (1988) Cell 55,849-856 20. Tsao, Y. P., Wu, H.-Y., and Liu, L. F. (1989) Cell 56, 111-118 21. Sekiguchi, M., and Iida, S. (1967) Proc. Natl. Acad. Sci. U. S. A.

58,2315-2320 22. Giaever, G. N., and Wang, J. C. (1988) Biophys. Chem. 29,7-15 23. Lodge, J. K., Kazic, T., and Berg, D. E. (1989) J. Bacterial. 171.

218i-2187 -.

24. Lockshon, D., and Morris, D. R. (1985) J. Mol. Biol. 181, 63-74 25. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular

Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

26. O’Connor, M. B., and Malamy, M. H. (1985) J. Mol. Biol. 181, 545-550

27. Gellert, M., Mizuuchi, K., O’Dea, M. H., Itoh, T., and Tomizawa, J. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 4772-4776

28. Sugino, A., Peebles, C. L., Kruezer, K. N., and Cozzarelli, N. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 4767-4771

29. Stuber, D., and Bujard, H. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,167-171

30. Pettijohn, D. E., and Pfenninger, 0. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 1331-1335

31. Drlica, K., and Rouviere-Yaniv, J. (1987) Microbial. Reu. 51, 301-319

32. Pettijohn, D. E. (1988) J. Biol. Chem. 263, 12793-12796 33. Baker, T. A., and Kornberg, A. (1988) Cell 55, 113-123 34. Mensa-Wilmont, K., Carroll, K., and McMacken, R. (1989)

EMBO J. 8, 2393-2402 35. Yang, L., Jessee, B. C., Lau, K., Zhang, H., and Liu, L. F. (1989)

Proc. Natl. Acad. Sci. U. S. A. 86,6121-6125