Aug 3, 1989 - ABSTRACT. We show that transcription originating at the. gyrA promoter of Escherichia coli is less subject to termination at the A T..p terminator ...
Proc. Natl. Acad. Sci. USA Vol. 86, pp. 8882-8886, November 1989 Genetics
The unexpected antitermination of gyrA-directed transcripts is enhanced by DNA relaxation (promoter/DNA supercoiling/gyrase/transcription/relaxation stimulation)
MAYNARD CARTY AND ROLF MENZEL E.I. du Pont de Nemours, Central Research and Development Division, Experimental Station, P.O. Box 80328, Wilmington, DE 19880-0328
Communicated by Martin Gellert, August 3, 1989
(pRAS101). A derivative of pRAS101 was then made in which the A Toop terminator intervenes between the gyrA promoter and lacZ (pPIC100). We observe termination with pPIC100 but find that it is less than expected. Even more surprising is the observation that termination decreases significantly upon DNA relaxation.
We show that transcription originating at the ABSTRACT gyrA promoter of Escherichia coli is less subject to termination at the A T..p terminator (22% read-through) than is transcription originating from either the galOP (1% read-through) or topA (3% read-through) control regions. Furthermore, termination of the gyrA transcripts decreases (60% read-through) upon relaxation of the template DNA. We believe that events related to stimulation of transcription at the gyrA promoter by DNA relaxation are responsible for the enhanced terminator read-through.
MATERIALS AND METHODS Strains and Plasmids. The plasmid pRAS101 has the EcoRI-BamHI gyrA promoter fragment of pRM345 (16) cloned into the promoterless Lac vector pMLB1109 (17). The plasmid pRAS102 has a 1-kilobase (kb) EcoRI fragment containing the galOP control region recloned from pKG1900 (18) and correctly oriented in pMLB1109. The plasmid pRAV2 has the EcoRI-BamHI fragment containing the topA promoters from the plasmid pBDR1 (19) ligated into pMLB1109. These three recombinant plasmids, and singlecopy A clone derivatives, will be described in more detail elsewhere (R.M. and R. Vargas, unpublished data). Plasmids containing the T0Op terminator were constructed in the course of this study as described in the legend to Fig. 1. All plasmids were transformed into the strain RFM 606 (20). DNA Manipulations. Restriction enzymes and T4 DNA ligase were purchased from either New England Biolabs or Bethesda Research Laboratories and used according to the vendor's recommendations. Dideoxy DNA sequencing was done using synthetic oligonucleotides and Sequenase (United States Biochemical) according to vendor specifications. Plasmid DNA was prepared by the cleared lysate method followed by two cycles of CsCI/ethidium bromide centrifugation (21). A plasmid (pKO1-T) containing the Top terminator on a 189-base-pair (bp) BamHI fragment was obtained from K. McKenney (National Bureau of Standards). The T,,0,p fragment was cleaved from the plasmid with BamHI and purified by gel electrophoresis (21). Growth Conditions. Culture media was prepared as described in Silhavey et al. (22). In experiment 1, Fig. 3, cultures were grown to stationary growth phase in M63 medium/0.3% fructose/20 ,ug of ampicillin per ml. These cultures were diluted 1:1000 into the same medium without ampicillin and grown to an OD6w value of between 0.2 and 0.4 before assay. Conditions for growth in the microtiter plates have been described elsewhere (20). ig-Galactosidase Assays. A modified f3-galactosidase assay was adapted from Miller (23) and scaled down to use microtiter plates as described (20). Final units are expressed as Miller units. In selected cases, activity was measured both as described by Miller and with our microtiter format; identical results were obtained. We have considered the possibility that the coumermycin treatment may induce cell shape changes that might be
The transcription of prokaryotic genes can be influenced by changes in DNA supercoiling (ref. 1; for review, see refs. 2 and 3) with a large fraction of promoters showing some response (4, 5). Transcription can be stimulated or reduced or remain unaffected by treatments that decrease DNA supercoiling. DNA supercoiling has been reported to change in response to temperature (6) and growth conditions (7, 8). It is tempting to speculate that DNA supercoiling can provide a global signal for coordinate changes in gene expression. This has been suggested for anaerobic growth (9, 10) and osmolarity changes (11). DNA supercoiling itself appears to be regulated in a homeostatic manner (12). Decreases in DNA supercoiling result in an increase in the expression of gyrA and gyrB, the genes encoding Escherichia coli's major DNA supercoiling activity (12), while effecting a decrease in the expression of topA, the gene encoding the cell's major DNA relaxing activity (13). In addition to presenting some intriguing possibilities for control strategies, changes in gene expression resulting from changes in DNA supercoiling also raise questions about the mechanisms effecting those changes. For promoters that are inactivated as a consequence of DNA relaxation, an intuitively appealing mechanism presents itself. Negative DNA supercoiling favors unwinding of the DNA double helix, and one could expect it to increase the rate of transcription for those promoters where open complex formation is ratelimiting. Experiments with purified RNA polymerase and supercoiled DNA have shown this effect (14, 15). The cases where transcription is enhanced by decreasing DNA supercoiling do not have such an easy explanation. A deletion analysis of gyrA and gyrB has implicated sequences extending from the -10 consensus hexamer of the promoter to the first few bases of the gyrase transcripts (16). In the gyrase examples it was suggested that relaxation-stimulated transcription (RST) involves promoter clearance-namely, events subsequent to polymerization of the first two nucleotides. We have constructed a plasmid derivative of pMLB1109 (17) in which the gyrA promoter directs transcription of lacZ The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: RST, relaxation-stimulated transcription.
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Proc. Natl. Acad. Sci. USA 86 (1989)
reflected in altered cell OD values. Examination of a culture by microscope following 2 hr of coumermycin treatment reveals no extensive changes. Much longer (>8 hr) treatments result in extensive filamentation typical of an SOS response. Induction values based on normalization to cell protein determined by Coomassie dye binding (Bio-Rad) are identical to those seen when normalization is made to cell OD. S-30 in Vitro Transcription Translation Protocols. S-30 in vitro transcription-translation reactions were done essentially as described by Jovanovich et al. (24). Either the strain JV 554 (24) or RFM 443 (20) was grown as a source of the S-30 extract. The A-galactosidase activity synthesized in the S-30 reaction mix was calculated and expressed as nmol of onitrophenyl P-galactoside hydrolyzed per minute by 25 al of the S-30 reaction mix.
RESULTS
To examine systematically the behavior of the A T00p terminator we constructed a set of plasmids with T,,0,p cloned between a promoter (either the gyrA, galOP, or topA promoter) and the reporter gene lacZ (Fig. 1). These plasmids
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were transformed into RFM 606 and monomeric forms of the constructs were isolated. Differences between these three systems deserve further consideration. In Fig. 2 we have illustrated the transcriptional and translational organization of our three experimental systems and that for T0,p in its native context. The gyrA system is notable for the short untranslated leader 5' of the terminator. In the gyrA case, one could imagine that the short distance between the promoter and terminator is insufficient for the loading of factors important to termination. Published experiments address this issue with Tarp in its natural context (26). In these earlier studies it was shown that T.,,, terminates well (80%) without Rho protein, and very efficiently (>99%) with Rho, indicating that the short leader is sufficient. Our artificial gyrA-T0p construct has a leader 13 bases longer than the native T00p transcript. The gyrA-terminator construct is in an untranslated region and we anticipate maximum termination since it has been shown that translation in the region of a terminator decreases termination for Rho-dependent (27) and Rhoindependent (28) terminators. Everything we know suggests effective terminator utilization in the gyrA-TOp construct. Before considering the details of terminator behavior we must point out some unexpected aspects of RST in our lac fusions. We measure the magnitude of the RST effect by an induction ratio defined as the activity in a gyraseinhibitor-treated experiment divided by the activity of a parallel untreated control. The data in Fig. 3 for pRAS101 show that induction of gyrA varies from 3.4 to 4.3 depending on the growth and induction protocol used. Curiously we also see induction in the galOP and the topA promoters (2.4 and 1.7, respectively). These observations contradict results previously reported for the galOP (5, 13, 16) and topA (13, 19) promoters in studies involving fusions to the galK gene. In the galK fusions, the galOP control sequences were neutral with respect to coumermycin treatment (induction ratio = 1.0), whereas the transcription directed by topA was inhibited by coumermycin treatment (induction ratio < 1.0). We believe (R. Vargas and R.M., unpublished data) that the relative magnitude of RST is a function of the promoter initiating transcription and the specific reporter gene being tran-
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galOP (pRAS102 and pPIC120) and topA (pRAV2 and pPIC140) plasmid constructs. We have also examined termination efficiency in singlecopy versions of the gyrA, galOP, and topA fusions (data not shown). A phage carrying the Lac fusions were isolated by recombination between the fusion plasmids and the A RZ5, and single lysogens were isolated using published procedures (17, 29). Only minor discrepancies are found between the plasmid and A results, and the general pattern is the same: only the gyrA transcripts give notable terminator readthrough. Read-through of the gyrA-initiated transcripts is once again increased following coumermycin treatment to the point where read-through is greater than termination. As a consequence, the fusion strains containing a single-copy gyrA-terminator construct (present as a A lysogen) also show RST values of up to 12-fold. In Vitro Studies. We are able to confirm our basic result in an S-30 transcription-translation system provided we carefully choose the template DNA concentration. In Fig. 4 Upper (in the experiment with the RFM 443 extract at 25 Ag of DNA per ml) we see that the gyrA promoter's 3.6-fold RST effect is amplified into a 10-fold effect in the TO.p containing construct pPIC100. At this DNA concentration the topA promoters show no RST induction and no measurable terminator read-through. The major difference between this in vitro experiment and the in vivo work is a conspicuous decrease in terminator read-through. Despite this reduction in absolute terminator read-through, the presence of the gyrase inhibitor (novobiocin) increases read-through in a manner similar to that seen in vivo, resulting in the amplified RST signal. In other experiments we find that the magnitude of the RST effect is a function of the DNA concentration in the S-30 extract. For gyrA-promoted P-galactosidase production, RST is between 10- and 25-fold at low DNA (100 ,ug/ml) the addition of the gyrase inhibitor has little or no affect (unpublished data). We believe variation in RST as a function of DNA concentration is indicative of the titration of factor(s) important to the RST response in the S-30. In this current study the system allows us to modulate the degree of RST by altering the DNA concentration. In Fig. 4 we show a representative set of assays for the gyrA and topA fusions. Included are assays done with two different S-30 preparations that differ somewhat in their behavior; the RFM 443 extract is more inducible but less active than the JV 554 extract. The results show that for the gyrA promoter at high DNA concentrations (100 ,ug/ml in the JV 554 extract) RST becomes minimal with a concomitant decrease in termination efficiency. In other experiments we see the same trend for a set of observations made with the RFM 443 extract (data not given). There is a correlation between RST and terminator efficiency for the gyrA promoter when we modulate RST by varying the DNA concentration in the S-30 system. This effect is opposite that seen in vivo when RST was varied as a function of the promoter directing transcription. There is, however, no compelling reason to believe that RST and terminator efficiency should change in the same manner when variation is the consequence of altering two dissimilar parameters (the promoter directing transcription and the DNA concentration). We believe that these different forms of correlation strengthen the argument that RST and antitermination are related and may indeed provide insight into the mechanism when better understood. The relationship between RST and antitermination can be seen in the correlation between the degrees of RST seen in the promoter and promoter-terminator construct pairs. At first glance this relationship might seem trivial. If the promoter is responding to DNA relaxation, one of course expects the
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Proc. Natl. Acad. Sci. USA 86 (1989)
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FIG. 4. DNA from plasmids pRAS101, pPIC100, pRAV2, and pPIC140 was prepared, and the indicated amounts of DNA were added to either S-30 extract RFM 443 or JV 554. We note that at the highest DNA concentration (100 ,Lg/ml) the system is saturated with respect to the capacity to synthesize more protein. However, the extracts are not saturated with respect to RNA synthesis capacity or topoisomerase activity. DNA concentrations of 10 ,tg/ml and 100 ,4g/ml are comparable in the rate and extent of their relaxation in response to novobiocin. The S-30 reactions were run in the absence (-novo) or presence (+novo: RFM 443, 75 ug/ml; JV 554, 4.8 Ag/ml; the respective optima for novobiocin stimulation in the two S-30 preparations) of novobiocin, and the level of 3-galactosidase activity produced was determined. Activity units are given in Upper for each of the reaction conditions. Percent termination (%term.) was calculated for the terminator in the gyrA and galOP plasmid sets as described in the legend to Fig. 3. (Lower) Graphic representation of the % read-through, also defined in the legend to Fig. 3.
terminated construct with the same promoter to undergo a similar response. To consider effects at the terminator we must first factor out the promoter RST. We do this by dividing the terminated RST by the unterminated RST and define the resulting value as the "terminator effect." In Fig. 5 we have plotted the value of the terminator effect against that of the promoter RST. The cumulative data of several experiments with different S-30 preparations, including the results with the gyrA and topA plasmid sets, can be approximated by a simple logarithmic function given by the line shown in Fig. 5. The promoter RST appears to be a reasonable predictor of the terminator effect. As the RST values increase at low DNA concentrations with the gyrA promoter, there is a corresponding increase in the terminator effect. The general relationship even seems to hold when a promoter is inhibited by novobiocin treatment (RST < 1) and the terminator shows greater activity (terminator effect < 1) as seen with the topA plasmid set at the 100 /xg of DNA per ml level.
FIG. 5. Relationship between events occurring at the promoter and subsequent events occurring at the terminator. As an example of the calculations for this plot, consider the gyrA set (pRAS101 and pPIC100) of the RFM 443 S-30 reactions at 25 ,tg of DNA per ml. In Fig. 4 we have (54 units for pRAS101, +novo)/(15 units for pRAS101, -novo) or an RST of 3.6 for the unterminated gyrA construct and, similarly (3.1/0.31), or a composite RST of 10 for pPIC100, the terminated construct, yielding a terminator effect of 2.8 (10/3.6). The analogous calculations were made for the gyrA series in the JV 554 extracts at 10 and 100 t&g of DNA per ml and for the topA series at 100 ,tg/ml in Fig. 4. Additional data points were calculated from other experiments similar to those in Fig. 4 (primary data not given). *, gyrA calculations; *, topA.
We have noted a similar relationship in vivo (although over a narrower range of values) where we can manipulate RST and terminator effect as a function of growth conditions (R.M., unpublished observations; limited data in Fig. 3).
DISCUSSION We have shown that the level of termination at TOOP is a function of the sequences directing transcription. Although the structure of the gyrA-Toop fusion (Fig. 2 and the accompanying text) suggests that the gyrA-directed transcripts should be subject to strong termination, we find they are significantly more able to read through the terminator than are topA- or galOP-directed transcription units. Termination efficiency seems to be a function of the sequences directing transcription. This is contrary to the generally accepted view that a terminator functions as an autonomous unit with an intrinsic termination efficiency (30) but is in agreement with recent results from the laboratories of Telesnitsky and Chamberlin (31) and Roberts and colleagues (32). We do not know if the antitermination property of the gyrA transcripts applies to termination signals other than A To0p. We also note that read-through of TOOP is enhanced by coumermycin treatment in the gyrA system. This is opposite to the finding of Rosenthal and Calvo in the leu system (33). Coumermycin treatment inhibits DNA gyrase resulting in DNA relaxation and the consequent induction of a relaxationstimulated (RST) class of promoters (5). The increase in promoter expression together with the decrease in termination efficiency gives us the amplified RST effect of the gyrA-TOp fusion constructs. We are able to reproduce read-through of T0Op by gyrAdirected transcripts, as well as enhancement of that readthrough, in a variety of experimental systems. The basic phenomenon can be demonstrated with plasmid and singlecopy A lysogen versions of the construct. The effect can also be shown in an S-30 extract provided that the DNA concentration is carefully chosen. The galOP and topA set provides controls in all experimental systems. We believe that the RST
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nature of the gyrA promoter is related to the ability of gyrA transcripts to read through TOOP. The gyrA promoter is the only promoter in our test set to exhibit a strong RST effect and the only promoter to give substantial read-through of TOOP. Further, when we induce the gyrA promoter with coumermycin we note a marked increase in the fraction of the transcripts that proceed past the terminator. Although we are able to reproduce our basic result in the S-30 system, to do so we must carefully choose our DNA concentration. RST is a function of the DNA concentration in the extract; at low DNA concentrations we see maximum RST, whereas at high DNA the effect diminishes. We find we are able to "titrate" the RST effect (increase expression from gyrA while reducing RST) by adding a second heterologous DNA and believe that this behavior represents the titration of a negative factor central to the RST effect (unpublished data). We hypothesize that this factor acts by reducing expression at low DNA concentrations in a manner reversible by DNA relaxation. At high DNA, when the factor has been titrated, RST is minimal. At low DNA concentrations where RST is apparent (for gyrA), we find larger composite RST values for the gyrA-TOOP constructs. When we increase the DNA concentration of the S-30, RST is no longer apparent and DNA relaxation no longer increases terminator read-through. The two effects, RST and terminator read-through, are related as shown in Fig. 5. This relationship between RST and increased terminator read-through implies that a reduction in the activity of our hypothesized negative factor leads to greater terminator read-through. If this is the case, we expect to see the absolute level of terminator read-through to increase at high DNA concentrations. This predicted increase in read-through at high DNA levels is seen in the S-30 experiments (2.1% increase to 14%). In addition to confirming the pattern of our in vivo observations at a carefully chosen DNA concentration, we find additional evidence of a relationship between RST and antitermination by inspecting the behavior of the S-30 under conditions where the in vivo results are not reproduced. Telesnitsky and Chamberlin (31) and Roberts and colleagues (32) have shown that the initial bases of a transcript are able to direct RNA polymerase into an antitermination mode. These bases overlap the region we believe is involved in the promoter clearance barrier that determines the RST character of the gyrase promoters (16). Although the work from Chamberlin and Roberts deals with factor-independent antitermination, other experiments demonstrate the interaction of the antitermination factor Q with the same region in the A PR promoter (34). We believe the results in this paper tell us that RST at the gyrA promoter is likely to involve termination and antitermination factors. We are indebted to Dr. Stevan Jovanovich for the JV 554 extract and for his patience in teaching us the preparation of S-30 extracts. 1. Sanzey, B. (1979) J. Bacteriol. 138, 40-47. 2. Gellert, M. (1981) Annu. Rev. Biochem. 50, 879-910.
Proc. Natl. Acad. Sci. USA 86 (1989) 3. Drlica, K. (1984) Microbiol. Rev. 48, 273-289. 4. Jovanovich, S. B. & Lebowitz, J. (1986) J. Bacteriol. 169, 4431-4435. 5. Menzel, R. & Gellert, M. (1986) J. Bacteriol. 169, 1272-1278. 6. Goldstein, E. & Drlica, K. (1984) Proc. Natl. Acad. Sci. USA 81, 4046-4050. 7. Shure, M., Pulleyblank, D. E. & Vinograd, J. (1977) Nucleic Acids Res. 4, 1183-1205. 8. Blake, V. L. & Gralla, J. D. (1987) J. Bacteriol. 169, 44994506. 9. Yamamoto, N. & Droffner, M. (1985) Proc. Natl. Acad. Sci. USA 83, 6805-6809. 10. Axley, M. J. & Stadtman, T. C. (1988) Proc. Natl. Acad. Sci. USA 85, 1023-1027. 11. Higgins, C. F., Dorman, C. J., Stirling, D. A., Waddell, L., Booth, I. R., May, G. & Bremer, E. (1988) Cell 52, 569-584. 12. Menzel, R. & Gellert, M. (1983) Cell 34, 105-113. 13. Tse-Dinh, Y.-C. (1985) Nucleic Acids Res. 13, 4751-4763. 14. Borowiec, J. A. & Gralla, J. D. (1985) J. Mol. Biol. 184, 587-598. 15. Wood, D. C. & Lebowitz, J. (1984) J. Biol. Chem. 259, 1118411187. 16. Menzel, R. & Gellert, M. (1987) Proc. Natl. Acad. Sci. USA 84,
4185-4189. 17. Roland, K. L., Liu, C. & Turnbough, C. L. (1988) Proc. Natl. Acad. Sci. USA 85, 7149-7153. 18. McKenney, K. H., Shimatake, H., Court, D., Schmeissner, U., Brady, C. & Rosenberg, M. (1981) in Structural Analysis of Nucleic Acids, eds. Chirikjian, J. A. & Papas, T. S. (Elsevier, New York), Vol. 2, pp. 383-415. 19. Tse-Dinh, Y.-C. & Beran, R. K. (1988) J. Mol. Biol. 202, 735-742. 20. Menzel, R. (1989) Anal. Biochem. 181, 40-50. 21. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 22. Silhavy, T., Berman, M. L. & Enquist, L. W. (1984) Experiments with Gene Fusions (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 23. Miller, J. H. (1972) Experiments with Molecular Genetics (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 24. Jovanovich, S. B., Lesley, S. A. & Burgess, R. R. (1989) J. Biot. Chem. 264, 3794-3799. 25. Schwartz, E., Scherer, G., Hobom, G. & Kossel, H. (1978) Nature (London) 272, 410-414. 26. Howard, B., de Crombrugghe, B. & Rosenberg, M. (1977) Nucleic Acids Res. 4, 827-842. 27. Platt, T. & Bear, D. G. (1983) in Gene Function in Procaryotes, eds. Beckwith, J., Davies, J. & Gallant, J. A. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 123-161. 28. Wright, J. J. & Hayward, R. S. (1987) EMBO J. 6, 1115-1119. 29. Simons, R. W., Houman, F. & Kleckner, N. (1987) 53, 85-96. 30. Rosenberg, M., McKenney, K. & Schumperli, D. (1982) in Promoters: Structure and Function, eds. Rodriguez, R. & Chamberlin, M. (Praeger, New York), pp. 387-406. 31. Telesnitsky, A. P. W. & Chamberlin, M. J. (1989) J. Mol. Biol. 205, 315-330. 32. Goliger, J. A., Yang, X., Guo, H.-C. & Roberts, J. W. (1989) J. Mol. Biol. 205, 331-341. 33. Rosenthal, E. R. & Calvo, J. M. (1987) Mol. Gen. Genet. 207, 430-434. 34. Grayhack, E. J., Yang, X. & Roberts, J. W. (1985) Cell 42, 259-269.
