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Monovalent cations differ in their effects on transcription initiation from a σ-70 promoter of Escherichia coli
Gene 196 ( 1997) 95–98
Monovalent cations dier in their eects on transcription initiation from a s-70 promoter of Escherichia coli Jian-Ying Wang a,b, Karl Drlica b, Michael Syvanen a,* a Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA 95616, USA b Public Health Research Institute, 455 First Avenue, New York, NY 10016, USA Received 27 September 1996; accepted 4 March 1997; Received by C.M. Kane
Abstract Initiation of transcription from the s-70 rep promoter of plasmid pBR322 was measured by abortive transcription assays at various concentrations of potassium, rubidium, and sodium acetate. When linear and negatively supercoiled templates were compared, each salt generated a characteristic response. Increasing the salt concentration decreased transcription from a linear template but produced an increase (potassium) or a bell-shaped response (rubidium) with a supercoiled template. In the case of sodium ions, increasing concentration inhibited transcription initiation from both linear and supercoiled templates. These results are discussed with respect to eects of monovalent cations on DNA twist. © 1997 Elsevier Science B.V. Keywords: DNA supercoiling; Potassium; Rubidium; Sodium
1. Introduction
2. Experimental
As part of an eort to understand control of gene expression, there have been many studies concerning how salt, temperature, and DNA conformation aect initiation of transcription (for an early review, see Losick and Chamberlin, 1976 ). Among the less understood phenomena are the bell-shaped responses of transcription initiation when potassium ion concentation or negative supercoiling are increased (Borowiec and Gralla, 1985; Mulligan et al., 1985; Brahms et al., 1985; Borowiec and Gralla, 1987; Prince and Villarejo, 1990). While examining the combined eect of monovalent cations and supercoiling on transcription initiation, we were surprised to find that dierent monovalent cations have distinctive eects on linear and supercoiled templates within the same concentration range. These observations, which we report below, are not completely explained by conventional considerations involving duplex stability and enzyme inhibition; thus an alternate idea involving DNA twist is also discussed.
To monitor initiation of transcription we performed abortive initiation assays ( McClure, 1985 ) using the rep promoter of plasmid pBR322 (Tomizawa, 1990; TTGAAG TGG TGG CCT AAC TAC GGC TACACT AGA AGG ACA GTA TTT GGT AT, in which the −35 region, the −10 region and the +1 nucleotide, respectively, are underlined ). RNA polymerase was first incubated with DNA templates to allow formation of heparin-resistant, ‘‘open’’ complexes. When a-32P ATP and the dinucleotide ApC were subsequently added to reactions, the enzyme–promoter complex catalysed the formation of ApC32pA. ApC32pA was quantified following gel electrophoresis (Fig. 1 ) as a measure of transcription initiation. Several lines of evidence confirmed that the band shown in Fig. 1 was ApC32pA and a transcription product of P ( Wood and Leibowitz, rep 1984; Brahms et al., 1985). First, the product of the reaction was resistant to phosphatase and sensitive to RNase A, which attacks the 3∞ side of a pyrimidine. Second, the reaction required ATP, and a-32P UTP would not substitute for a-32P ATP. Third, the product band disappeared when the other three nucleoside triphosphates were incubated along with a-32P-ATP, pre-
* Corresponding author. Tel: +1 916 7524991; Fax: +1 916 7528692; e-mail: [email protected] Abbreviations: EDTA, ethylenediamine tetraacetic acid 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0 3 78 - 11 19 ( 9 7 ) 00 20 7 -2
96
J.-Y. Wang et al. / Gene 196 (1997) 95–98
Fig. 1. Assay for eects of monovalent cations on initiation of transcription. Abortive transcription was carried out as described ( Borowiec and Gralla, 1985 ). DNA ( 2.4 nM) in 30 mM Tris-acetate ( pH 8.0), 3 mM magnesium acetate, 0.2 mM dithiothreitol, 0.1 mM EDTA, 100 mg/ml bovine serum albumin, 3.25% (v/v) glycerol, and the indicated concentration of potassium acetate was incubated at 37°C for 2 min. E. coli RNA polymerase ( U.S. Biochemical Corp; 0.007 units; 8.5-fold molar excess relative to DNA) in the same buer at 37°C was then added to allow open complex formation. After an appropriate time interval, a 10 ml sample was withdrawn and mixed with 5 ml ApC (Sigma Chemical Corp; 400 mM final concentration) and [a-32P ]ATP (10 mM, 10 Ci/mmol ), 30 mM Tris-acetate (pH 8.0 ), 3 mM magnesium acetate, 0.2 mM dithiothreitol, 0.1 mM EDTA, and heparin ( 100 mg/ml ) to initiate abortive transcription. Reactions were stopped by addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol. Ten ml aliquots were subjected to electrophoresis in a 7 M urea–8% polyacrylamide gel. Shown is an autoradiogram of the trinucleotide, ApC32pA produced from P after 30 min incubation at the indicated rep concentrations of potassium acetate for linear ( A) or negatively supercoiled ( B) DNA templates (only one band was visible on gels other than unincorporated ATP). The production of ApCpA was linear between 0 and 30 min (data not shown). The amount of ApCpA produced after 30 min in 0.04 M potassium acetate was 166 nmol and 92 nmol of trinucleotide per nmol of the relaxed and supercoiled DNA templates, respectively.
sumably because the ApCpA product was extended to a long transcript. Fourth, digestion of pBR322 with HindIII, PstI and PvuII gave three DNA fragments, each of which was tested by the abortive initiation assay. Only the PstI–PvuII fragment, which contains P , gave rep rise to the band. The eect of potassium acetate on initiation of transcription from linear and supercoiled templates is shown in Figs. 1 and 2A. Increasing potassium concentration inhibited transcription from linear pBR322 but stimulated it from negatively supercoiled plasmid. With respect to absolute activity at high potassium concentration (0.44 M ), we observed no transcription activity (<1 pmol product per pmol template) for linear DNA while 100 pmol were produced per pmol template from the negatively supercoiled template (data not shown).
Fig. 2. Eect of monovalent ions on initiation of transcription. Reactions were carried out as described in the legend to Fig. 1 at the indicated monovalent salt concentrations using linear ($–$) or negatively supercoiled (#–#; s=−0.04 ) templates. Autoradiograms similar to those shown in Fig. 1 were obtained, and the X-ray film was scanned using a Molecular Dynamic Scanner with a laser light source to measure the relative quantity of abortive transcription products. The measured darkening of the film was linear with added 32P between 300 and 50 000 cpm. Linearity was assured by adjusting the exposure times of the film. Salts added to reaction mixtures were potassium acetate ( A), rubidium acetate (B) and sodium acetate (C ). Rubidium acetate is extremely hygroscopic. A defined concentration was prepared from a powder that had been dried in a vacuum oven for 24 h at 90°C, cooled under vacuum and immediately weighed.
Thus, in very high salt, supercoiling of pBR322 activates P by at least a factor of 100 compared with its linear rep form. Supercoiling clearly overcomes the inhibitory eect of potassium seen with linear templates. Since multiple trinucleotides are synthesized for each enzyme–promoter complex (McClure, 1985 ), the reaction monitored is an idling reaction in which the amount
J.-Y. Wang et al. / Gene 196 (1997) 95–98
of trinucleotide produced is an indirect measure of both open complex concentration and turnover number. We found that the more than 100-fold decline in activity for the linear template was due largely to inhibition of open complex formation rather than the idling reaction, since when the heparin-resistant complex was allowed to form in 0.04 M potassium acetate followed by performing the idling reaction at 0.44 M, the amount of trinucleotide product was still half that observed when both steps were carried out at 0.04 M (data not shown). Thus, raising the potassium ion concentration had a minor eect on the idling reaction compared with its eect on the overall reaction with the linear template. When we examined the eect of rubidium acetate on transcription initiation from both templates, we found that increasing the rubidium concentration inhibited transcription from the linear template, but transcription was stimulated at concentrations of up to 0.3 M from supercoiled DNA (Fig. 2B). Higher rubidium concentrations reduced transcription activity (Fig. 2B). When sodium acetate was tested with the two templates, activity decreased in both cases ( Fig. 2C ). These results raise two questions: (1) why does increasing K+ and Rb+ stimulate transcription from the supercoiled template while inhibiting it from the linear template?; and (2 ) why does Na+ behave so dierently? Several possibilities are considered below.
3. Discussion Monovalent cations are thought to have a variety of eects on transcription initiation. For example, they raise DNA melting temperature, and that should make open complex formation require more energy at high salt concentration (Ohlsen and Gralla, 1992). Adding salt also adversely aects the displacement of cations that occurs when protein binds to DNA ( Roe and Record, 1985). That is expected to interfere with RNA polymerase binding to DNA. Salt may even directly inhibit RNA polymerase activity (Prince and Villarejo, 1990 ). It is also possible that supercoiled DNA is under much greater torsional stress at very low salt conditions than at high ones (Hunt and Hearst, 1991), although even the lowest salt concentrations used in the present study would all be considered high with respect to torsional stress and would probably not show much dierence. Any of these eects could explain why increasing sodium ion lowers transcription from both linear and supercoiled templates. The eects could also produce the declining phase of the bell-shaped curve seen with rubidium (Fig. 2B). The ascending phase, which is restricted to supercoiled DNA and is seen with potassium and rubidium but not sodium (Fig. 2 ), could be explained by supercoiling causing exposure of singlestranded DNA that results in non-productive RNA
97
polymerase–DNA complexes, which might then be dissociated by increasing salt. To explain the dierence between sodium and the other two cations, one might postulate that sodium has a particularly toxic eect on proteins. However, in the present case potassium and rubidium ions inhibit transcription from the linear template at least as much as does sodium ( Fig. 2). Thus another explanation for the salt dierences is necessary. Wang and Syvanen (1992) proposed that when RNA polymerase binds to two elements on DNA (the −35 and −10 regions of promoters), the relative position of those elements, with respect to axial rotation, can be altered by DNA twist such that twist-altering factors would influence the ability of RNA polymerase to interact with the promoter. Since increasing potassium and rubidium ion concentration causes DNA to positively twist (Anderson and Bauer, 1978 ) and negative supercoiling lowers DNA twist (Borowiec and Gralla, 1987), supercoiled DNA would require higher potassium and rubidium concentrations to achieve the same level of DNA twist as found in a linear template. Thus, if in low salt the promoter in a supercoiled template is undertwisted, then potassium and rubidium ions would stimulate activity by restoring optimal orientation of the −10 and −35 regions. In contrast, linear templates, which are more twisted than their supercoiled counterparts, may already be at or above the optimal twist for the promoter; thus, increasing the twist by raising potassium or rubidium concentration would not stimulate transcription. Indeed, further twisting could contribute to the decline in activity seen in Fig. 2A and B as high salt concentrations would rotate the two regions out of the optimal orientation. Since DNA twist is greater in a rubidium buer than in a potassium buer (Anderson and Bauer, 1978), less rubidium than potassium would be required to achieve an optimum. Rubidium also causes a higher incremental change in twist upon changing salt concentration, so rubidium eects should cause a sharper peak of transcription initiation than potassium when the template is supercoiled. In the present case, the peak due to potassium is so broad that there is only a hint of an optimum at the highest concentrations tested (Fig. 2A), while that due to rubidium is quite distinct ( Fig. 2B). Sodium has a much smaller eect on DNA twist than does either potassium or rubidium. Indeed, a concentration of sodium ion higher than 10 M would be required to induce the same degree of twist change as 0.34 M potassium ion (Anderson and Bauer, 1978). This is far beyond conditions normally used for abortive transcription initiation. Therefore, within the salt range explored with potassium and rubidium, sodium should display little or no twist-related eect on transcription initiation. The relative orientation of the −10 and −35 regions is also influenced by spacer length, the number of nucleotides separating the two regions. For most E. coli
98
J.-Y. Wang et al. / Gene 196 (1997) 95–98
promoters, the spacer length is 17 bp. In the present case, the spacer length is 18 bp, and a transcription optimum was observed at moderate concentrations of rubidium ( Fig. 2B). According to the twist-transcription hypothesis, shorter spacers would require higher salt concentrations to achieve optimal transcription initiation. This prediction is now being tested. While the twist hypothesis is conceptually simple, changes in twist have not been correlated with changes in transcription activity, largely because direct measurement of twist is experimentally dicult. Eorts are underway to overcome this technical problem.
Acknowledgement This work was supported by a grant from the U.S. Army awarded to M.S.; K.D. was supported by NIH grant AI 35257. We thank Tom Record for critically reading and contributing to this paper.
References Anderson, P., Bauer, W., 1978. Supercoiling in closed circular DNA: dependence upon ion type and concentration. Biochemistry 17, 594–601.
Borowiec, J.A., Gralla, J.D., 1985. Supercoiling response of the lac ps promoter in vitro. J. Mol. Biol. 184, 587–598. Borowiec, J.A., Gralla, J.D., 1987. All three elements of the lac ps promoter mediate its transcriptional response to DNA supercoiling. J. Mol. Biol. 195, 89–97. Brahms, J., Dargouge, O., Brahms, S., Ohara, Y., Vagner, V., 1985. Activation and inhibition of transcription by supercoiling. J. Mol. Biol. 181, 455–465. Hunt, N.G., Hearst, J.E., 1991. Elastic model of DNA supercoiling in the infinite-length limit. J. Chem. Physics 95, 9329–9336. Losick, R., Chamberlin, M., (Eds.) 1976. RNA Polymerase. Cold Spring Harbor Press, Cold Spring Harbor, NY. McClure, W.R., 1985. Mechanism and control of transcription initiation in prokaryotes. Annu. Rev. Biochem. 54, 171–204. Mulligan, M.E., Brosius, J., McClure, W.R., 1985. Characterization in vitro of the eect of spacer length on the activity of Escherichia coli RNA polymerase at the tac promoter. J. Biol. Chem. 260, 3529–3538. Ohlsen, K.L., Gralla, J.D., 1992. Interrelated eects of DNA supercoiling, ppGpp, and low salt on melting within the Escherichia coli ribosomal RNA rrnB P1 promoter. Mol. Microbiol. 6, 2243–2251. Prince, W.S., Villarejo, M.R., 1990. Osmotic control of proU transcription is mediated through direct action of potassium glutamate on the transcription complex. J. Biol. Chem. 265, 17673–17679. Roe, J.H., Record, M.T., 1985. Regulation of the kinetics of the interaction of E. coli RNA polymerase with the lPR promoter by salt concentration. Biochemistry 24, 4721–4726. Tomizawa, J., 1990. Control of colE1 plasmid replication. J. Mol. Biol. 220, 683–694. Wang, J.-Y., Syvanen, M., 1992. DNA twist as a transcriptional sensor for environmental changes. Mol. Microbiol. 6, 1861–1866. Wood, D.C., Leibowitz, J., 1984. Eect of supercoiling on the abortive initiation kinetics of the RNA-I promoter of colE1 plasmid DNA. J. Biol. Chem. 259, 11184–11187.
Monovalent cations dier in their eects on transcription initiation from a s-70 promoter of Escherichia coli Jian-Ying Wang a,b, Karl Drlica b, Michael Syvanen a,* a Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA 95616, USA b Public Health Research Institute, 455 First Avenue, New York, NY 10016, USA Received 27 September 1996; accepted 4 March 1997; Received by C.M. Kane
Abstract Initiation of transcription from the s-70 rep promoter of plasmid pBR322 was measured by abortive transcription assays at various concentrations of potassium, rubidium, and sodium acetate. When linear and negatively supercoiled templates were compared, each salt generated a characteristic response. Increasing the salt concentration decreased transcription from a linear template but produced an increase (potassium) or a bell-shaped response (rubidium) with a supercoiled template. In the case of sodium ions, increasing concentration inhibited transcription initiation from both linear and supercoiled templates. These results are discussed with respect to eects of monovalent cations on DNA twist. © 1997 Elsevier Science B.V. Keywords: DNA supercoiling; Potassium; Rubidium; Sodium
1. Introduction
2. Experimental
As part of an eort to understand control of gene expression, there have been many studies concerning how salt, temperature, and DNA conformation aect initiation of transcription (for an early review, see Losick and Chamberlin, 1976 ). Among the less understood phenomena are the bell-shaped responses of transcription initiation when potassium ion concentation or negative supercoiling are increased (Borowiec and Gralla, 1985; Mulligan et al., 1985; Brahms et al., 1985; Borowiec and Gralla, 1987; Prince and Villarejo, 1990). While examining the combined eect of monovalent cations and supercoiling on transcription initiation, we were surprised to find that dierent monovalent cations have distinctive eects on linear and supercoiled templates within the same concentration range. These observations, which we report below, are not completely explained by conventional considerations involving duplex stability and enzyme inhibition; thus an alternate idea involving DNA twist is also discussed.
To monitor initiation of transcription we performed abortive initiation assays ( McClure, 1985 ) using the rep promoter of plasmid pBR322 (Tomizawa, 1990; TTGAAG TGG TGG CCT AAC TAC GGC TACACT AGA AGG ACA GTA TTT GGT AT, in which the −35 region, the −10 region and the +1 nucleotide, respectively, are underlined ). RNA polymerase was first incubated with DNA templates to allow formation of heparin-resistant, ‘‘open’’ complexes. When a-32P ATP and the dinucleotide ApC were subsequently added to reactions, the enzyme–promoter complex catalysed the formation of ApC32pA. ApC32pA was quantified following gel electrophoresis (Fig. 1 ) as a measure of transcription initiation. Several lines of evidence confirmed that the band shown in Fig. 1 was ApC32pA and a transcription product of P ( Wood and Leibowitz, rep 1984; Brahms et al., 1985). First, the product of the reaction was resistant to phosphatase and sensitive to RNase A, which attacks the 3∞ side of a pyrimidine. Second, the reaction required ATP, and a-32P UTP would not substitute for a-32P ATP. Third, the product band disappeared when the other three nucleoside triphosphates were incubated along with a-32P-ATP, pre-
* Corresponding author. Tel: +1 916 7524991; Fax: +1 916 7528692; e-mail: [email protected] Abbreviations: EDTA, ethylenediamine tetraacetic acid 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0 3 78 - 11 19 ( 9 7 ) 00 20 7 -2
96
J.-Y. Wang et al. / Gene 196 (1997) 95–98
Fig. 1. Assay for eects of monovalent cations on initiation of transcription. Abortive transcription was carried out as described ( Borowiec and Gralla, 1985 ). DNA ( 2.4 nM) in 30 mM Tris-acetate ( pH 8.0), 3 mM magnesium acetate, 0.2 mM dithiothreitol, 0.1 mM EDTA, 100 mg/ml bovine serum albumin, 3.25% (v/v) glycerol, and the indicated concentration of potassium acetate was incubated at 37°C for 2 min. E. coli RNA polymerase ( U.S. Biochemical Corp; 0.007 units; 8.5-fold molar excess relative to DNA) in the same buer at 37°C was then added to allow open complex formation. After an appropriate time interval, a 10 ml sample was withdrawn and mixed with 5 ml ApC (Sigma Chemical Corp; 400 mM final concentration) and [a-32P ]ATP (10 mM, 10 Ci/mmol ), 30 mM Tris-acetate (pH 8.0 ), 3 mM magnesium acetate, 0.2 mM dithiothreitol, 0.1 mM EDTA, and heparin ( 100 mg/ml ) to initiate abortive transcription. Reactions were stopped by addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol. Ten ml aliquots were subjected to electrophoresis in a 7 M urea–8% polyacrylamide gel. Shown is an autoradiogram of the trinucleotide, ApC32pA produced from P after 30 min incubation at the indicated rep concentrations of potassium acetate for linear ( A) or negatively supercoiled ( B) DNA templates (only one band was visible on gels other than unincorporated ATP). The production of ApCpA was linear between 0 and 30 min (data not shown). The amount of ApCpA produced after 30 min in 0.04 M potassium acetate was 166 nmol and 92 nmol of trinucleotide per nmol of the relaxed and supercoiled DNA templates, respectively.
sumably because the ApCpA product was extended to a long transcript. Fourth, digestion of pBR322 with HindIII, PstI and PvuII gave three DNA fragments, each of which was tested by the abortive initiation assay. Only the PstI–PvuII fragment, which contains P , gave rep rise to the band. The eect of potassium acetate on initiation of transcription from linear and supercoiled templates is shown in Figs. 1 and 2A. Increasing potassium concentration inhibited transcription from linear pBR322 but stimulated it from negatively supercoiled plasmid. With respect to absolute activity at high potassium concentration (0.44 M ), we observed no transcription activity (<1 pmol product per pmol template) for linear DNA while 100 pmol were produced per pmol template from the negatively supercoiled template (data not shown).
Fig. 2. Eect of monovalent ions on initiation of transcription. Reactions were carried out as described in the legend to Fig. 1 at the indicated monovalent salt concentrations using linear ($–$) or negatively supercoiled (#–#; s=−0.04 ) templates. Autoradiograms similar to those shown in Fig. 1 were obtained, and the X-ray film was scanned using a Molecular Dynamic Scanner with a laser light source to measure the relative quantity of abortive transcription products. The measured darkening of the film was linear with added 32P between 300 and 50 000 cpm. Linearity was assured by adjusting the exposure times of the film. Salts added to reaction mixtures were potassium acetate ( A), rubidium acetate (B) and sodium acetate (C ). Rubidium acetate is extremely hygroscopic. A defined concentration was prepared from a powder that had been dried in a vacuum oven for 24 h at 90°C, cooled under vacuum and immediately weighed.
Thus, in very high salt, supercoiling of pBR322 activates P by at least a factor of 100 compared with its linear rep form. Supercoiling clearly overcomes the inhibitory eect of potassium seen with linear templates. Since multiple trinucleotides are synthesized for each enzyme–promoter complex (McClure, 1985 ), the reaction monitored is an idling reaction in which the amount
J.-Y. Wang et al. / Gene 196 (1997) 95–98
of trinucleotide produced is an indirect measure of both open complex concentration and turnover number. We found that the more than 100-fold decline in activity for the linear template was due largely to inhibition of open complex formation rather than the idling reaction, since when the heparin-resistant complex was allowed to form in 0.04 M potassium acetate followed by performing the idling reaction at 0.44 M, the amount of trinucleotide product was still half that observed when both steps were carried out at 0.04 M (data not shown). Thus, raising the potassium ion concentration had a minor eect on the idling reaction compared with its eect on the overall reaction with the linear template. When we examined the eect of rubidium acetate on transcription initiation from both templates, we found that increasing the rubidium concentration inhibited transcription from the linear template, but transcription was stimulated at concentrations of up to 0.3 M from supercoiled DNA (Fig. 2B). Higher rubidium concentrations reduced transcription activity (Fig. 2B). When sodium acetate was tested with the two templates, activity decreased in both cases ( Fig. 2C ). These results raise two questions: (1) why does increasing K+ and Rb+ stimulate transcription from the supercoiled template while inhibiting it from the linear template?; and (2 ) why does Na+ behave so dierently? Several possibilities are considered below.
3. Discussion Monovalent cations are thought to have a variety of eects on transcription initiation. For example, they raise DNA melting temperature, and that should make open complex formation require more energy at high salt concentration (Ohlsen and Gralla, 1992). Adding salt also adversely aects the displacement of cations that occurs when protein binds to DNA ( Roe and Record, 1985). That is expected to interfere with RNA polymerase binding to DNA. Salt may even directly inhibit RNA polymerase activity (Prince and Villarejo, 1990 ). It is also possible that supercoiled DNA is under much greater torsional stress at very low salt conditions than at high ones (Hunt and Hearst, 1991), although even the lowest salt concentrations used in the present study would all be considered high with respect to torsional stress and would probably not show much dierence. Any of these eects could explain why increasing sodium ion lowers transcription from both linear and supercoiled templates. The eects could also produce the declining phase of the bell-shaped curve seen with rubidium (Fig. 2B). The ascending phase, which is restricted to supercoiled DNA and is seen with potassium and rubidium but not sodium (Fig. 2 ), could be explained by supercoiling causing exposure of singlestranded DNA that results in non-productive RNA
97
polymerase–DNA complexes, which might then be dissociated by increasing salt. To explain the dierence between sodium and the other two cations, one might postulate that sodium has a particularly toxic eect on proteins. However, in the present case potassium and rubidium ions inhibit transcription from the linear template at least as much as does sodium ( Fig. 2). Thus another explanation for the salt dierences is necessary. Wang and Syvanen (1992) proposed that when RNA polymerase binds to two elements on DNA (the −35 and −10 regions of promoters), the relative position of those elements, with respect to axial rotation, can be altered by DNA twist such that twist-altering factors would influence the ability of RNA polymerase to interact with the promoter. Since increasing potassium and rubidium ion concentration causes DNA to positively twist (Anderson and Bauer, 1978 ) and negative supercoiling lowers DNA twist (Borowiec and Gralla, 1987), supercoiled DNA would require higher potassium and rubidium concentrations to achieve the same level of DNA twist as found in a linear template. Thus, if in low salt the promoter in a supercoiled template is undertwisted, then potassium and rubidium ions would stimulate activity by restoring optimal orientation of the −10 and −35 regions. In contrast, linear templates, which are more twisted than their supercoiled counterparts, may already be at or above the optimal twist for the promoter; thus, increasing the twist by raising potassium or rubidium concentration would not stimulate transcription. Indeed, further twisting could contribute to the decline in activity seen in Fig. 2A and B as high salt concentrations would rotate the two regions out of the optimal orientation. Since DNA twist is greater in a rubidium buer than in a potassium buer (Anderson and Bauer, 1978), less rubidium than potassium would be required to achieve an optimum. Rubidium also causes a higher incremental change in twist upon changing salt concentration, so rubidium eects should cause a sharper peak of transcription initiation than potassium when the template is supercoiled. In the present case, the peak due to potassium is so broad that there is only a hint of an optimum at the highest concentrations tested (Fig. 2A), while that due to rubidium is quite distinct ( Fig. 2B). Sodium has a much smaller eect on DNA twist than does either potassium or rubidium. Indeed, a concentration of sodium ion higher than 10 M would be required to induce the same degree of twist change as 0.34 M potassium ion (Anderson and Bauer, 1978). This is far beyond conditions normally used for abortive transcription initiation. Therefore, within the salt range explored with potassium and rubidium, sodium should display little or no twist-related eect on transcription initiation. The relative orientation of the −10 and −35 regions is also influenced by spacer length, the number of nucleotides separating the two regions. For most E. coli
98
J.-Y. Wang et al. / Gene 196 (1997) 95–98
promoters, the spacer length is 17 bp. In the present case, the spacer length is 18 bp, and a transcription optimum was observed at moderate concentrations of rubidium ( Fig. 2B). According to the twist-transcription hypothesis, shorter spacers would require higher salt concentrations to achieve optimal transcription initiation. This prediction is now being tested. While the twist hypothesis is conceptually simple, changes in twist have not been correlated with changes in transcription activity, largely because direct measurement of twist is experimentally dicult. Eorts are underway to overcome this technical problem.
Acknowledgement This work was supported by a grant from the U.S. Army awarded to M.S.; K.D. was supported by NIH grant AI 35257. We thank Tom Record for critically reading and contributing to this paper.
References Anderson, P., Bauer, W., 1978. Supercoiling in closed circular DNA: dependence upon ion type and concentration. Biochemistry 17, 594–601.
Borowiec, J.A., Gralla, J.D., 1985. Supercoiling response of the lac ps promoter in vitro. J. Mol. Biol. 184, 587–598. Borowiec, J.A., Gralla, J.D., 1987. All three elements of the lac ps promoter mediate its transcriptional response to DNA supercoiling. J. Mol. Biol. 195, 89–97. Brahms, J., Dargouge, O., Brahms, S., Ohara, Y., Vagner, V., 1985. Activation and inhibition of transcription by supercoiling. J. Mol. Biol. 181, 455–465. Hunt, N.G., Hearst, J.E., 1991. Elastic model of DNA supercoiling in the infinite-length limit. J. Chem. Physics 95, 9329–9336. Losick, R., Chamberlin, M., (Eds.) 1976. RNA Polymerase. Cold Spring Harbor Press, Cold Spring Harbor, NY. McClure, W.R., 1985. Mechanism and control of transcription initiation in prokaryotes. Annu. Rev. Biochem. 54, 171–204. Mulligan, M.E., Brosius, J., McClure, W.R., 1985. Characterization in vitro of the eect of spacer length on the activity of Escherichia coli RNA polymerase at the tac promoter. J. Biol. Chem. 260, 3529–3538. Ohlsen, K.L., Gralla, J.D., 1992. Interrelated eects of DNA supercoiling, ppGpp, and low salt on melting within the Escherichia coli ribosomal RNA rrnB P1 promoter. Mol. Microbiol. 6, 2243–2251. Prince, W.S., Villarejo, M.R., 1990. Osmotic control of proU transcription is mediated through direct action of potassium glutamate on the transcription complex. J. Biol. Chem. 265, 17673–17679. Roe, J.H., Record, M.T., 1985. Regulation of the kinetics of the interaction of E. coli RNA polymerase with the lPR promoter by salt concentration. Biochemistry 24, 4721–4726. Tomizawa, J., 1990. Control of colE1 plasmid replication. J. Mol. Biol. 220, 683–694. Wang, J.-Y., Syvanen, M., 1992. DNA twist as a transcriptional sensor for environmental changes. Mol. Microbiol. 6, 1861–1866. Wood, D.C., Leibowitz, J., 1984. Eect of supercoiling on the abortive initiation kinetics of the RNA-I promoter of colE1 plasmid DNA. J. Biol. Chem. 259, 11184–11187.