Dependence of transcription-coupled DNA supercoiling on promoter strength in Escherichia coli topoisomerase I deficient strains

Abstract

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA in vitro and in vivo. This phenomenon has been nicely explained by a "twin-supercoiled-domain" model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In Escherichia coli topA strains, DNA gyrase selectively converts the positively supercoiled domain into negative supercoils to produce hypernegatively supercoiled DNA. In this article, in order to examine whether promoter strength affects transcription-coupled DNA supercoiling (TCDS), we developed a two-plasmid system in which a linear, non-supercoiled plasmid was used to express lac repressor constitutively while a circular plasmid was used to gage TCDS in E. coli cells. Using this two-plasmid system, we found that TCDS in topA strains is dependent on promoter strength. We also demonstrated that transcription-coupled hypernegative supercoiling of plasmid DNA did not need the expression of a membrane-insertion protein for strong promoters; however, it might require co-transcriptional synthesis of a polypeptide. Furthermore, we found that for weak promoters the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. Our results can be explained by the "twin-supercoiled-domain" model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA.

Figure 1. A two-plasmid system to study TCDS in vivo

This system contains two plasmids, a linear plasmid (A), i.e., pZXD51 and a circular, supercoiling-reporter plasmid (B), such as pZXD44. The linear plasmid is derived from linear coliphage N15-based plasmid pG591 and carries a laci gene under the control of the strong P_lacI^q promoter. E. coli cells containing pZXD51 over-express lac repressor (LacI) constitutively, which binds to the lac O1 operator (the open circle) on the supercoiling-reporter plasmids. The supercoiling-reporter plasmids were derived from plasmid pBR322 and constructed as detailed under Experimental Procedures. They harbor an IPTG-inducible promoter with different strengths and a transcription unit between the promoter and a set of 4 Rho-independent E. coli rrnB T1 terminators (winged triangles). (C)The DNA sequence of five different E. coli promoters P_T7A1/O4, P_tac, P_lacUV5, P_lac, and P_lacL8. The underlines represent the lac O1 operators.

Figure 2. RT-PCR analysis of cDNA products of mRNA transcribed from different supercoiling-reporter plasmids pZXD44, 50, 49, 47, and 48 in E. coli topA strain VS111 harboring the linear plasmid pZXD51 after 10 min of IPTG induction (1 mM)

(A) RT-PCR experiments were performed as described under Experimental Procedures. The lower panel is a 1.2% agarose gel containing 1% formaldehyde to show the integrity of the RNA samples used for the RT-PCR experiments. The upper panel is a 12% polyacrylamide gel in 1×TAE buffer to show the PCR products of 16S rRNA and tet gene cDNA synthesized from the RNA samples isolated from E. coli strain VS111 carrying supercoiling-reporter plasmids pZXD44, 50, 49, 47, and 48 after 10 min of IPTG induction (lanes 1–5 respectively). Labels: PR, promoter; T7A1, the T7A1/O4 promoter; tac, the tac promoter; lacUV5, the lacUV5 promoter; lac, the lac promoter; lacL8, the lacL8 promoter. (B) Real-time RT-PCR analyses of the tet gene mRNA for E. coli strain VS111 carrying different supercoiling-reporter plasmids pZXD44, 50, 49, 47, and 48 after 10 min of IPTG induction (mean±SD, three independent experiments). The relative level of RT-PCR products is proportional to the promoter strength. Promoter strength in P_bla units was obtained from ref.(Lanzer and Bujard 1988)

Figure 3. TCDS in E. coli strain VS111 is dependent on promoter strength

The in vivo T-S assays were performed as described under Experimental Procedures. DNA topoisomers were resolved by electrophoresis in a 1% agarose gel containing 2.5 µg/ml chloroquine and stained with ethidium bromide. The fastest moving band in the gels where DNA topoisomers are no longer resolvable under our experimental conditions represents the hypernegatively supercoiled DNA. (A) Dependence of TCDS on IPTG concentration for plasmid pZXD44 carrying a tet gene under the control of the strong P_T7A1/O4 promoter. Lane 1 contained the DNA sample isolated from E. coli cells prior to IPTG induction. Lanes 2–5 contained the DNA samples isolated from E. coli cells after 10 min of induction with 25, 50, 100, and 1000 µM IPTG, respectively. (B) Time course of the hypernegative supercoiling of plasmid pZXD44 in E. coli strain VS111 after 1 mM of IPTG induction. Lanes 1–4 contained, respectively, DNA samples isolated from VS111 after 0, 5, 10, and 15 min of IPTG induction. (C) Dependence of TCDS on promoter strength. Lanes 1–5 contained, respectively, DNA samples isolated from E. coli topA strain VS111 containing plasmids pZXD44, 50, 49, 47, and 48 after 5 min of IPTG (1 mM) induction. These plasmids carry a tet gene under the control of IPTG-inducible promoters with different strengths, i.e., promoters P_T7A1/O4, P_tac, P_lacUV5, P_lac, and P_lacL8. Labels: PR, promoter; T7A1, the T7A1/O4 promoter; tac, the tac promoter; lacUV5, the lacUV5 promoter; lac, the lac promoter; lacL8, the lacL8 promoter. (D) The percentage of hypernegatively supercoiled DNA is proportional to promoter strength (the values are the average of at least three independent determinations and the standard deviations are shown). These results were calculated as described under Experimental Procedures using the TCDS data shown in (C). Promoter strength in P_bla units was obtained from ref. (Lanzer and Bujard 1988)

Figure 4. For strong promoters, TCDS in E. coli topA strain VS111 did not require the expression of a membrane insertion protein

The in vivo T-S assays for plasmids carrying a GFP_UV gene under the control of IPTG-inducible promoters with different strengths were performed as described under Experimental Procedures. DNA topoisomers were resolved by electrophoresis in a 1% agarose gel containing 2.5 µg/ml chloroquine and stained with ethidium bromide. (A) and (C) The time course of the hypernegative supercoiling of plasmid pZXD57 carrying a GFP_UV gene under the control of the strong P_T7A1/O4 promoter in E. coli strain VS111 after 1 mM of IPTG induction. Lanes 1–4 contained, respectively, DNA samples isolated from VS111 after 0, 5, 10, and 15 min of IPTG induction. (B) and (D) Dependence of TCDS on promoter strength. Lanes 1–5 contained, respectively, DNA samples isolated from E. coli topA strain VS111 containing plasmids pZXD57, 58, 56, 55, and 54, that carry a cytosolic GFPuv gene under the control of IPTG-inducible promoters with different strengths, i.e., promoters P_T7A1/O4, P_tac, P_lacUV5, P_lac, and P_lacL8 after 5 min of IPTG (1 mM) induction. Labels: PR, promoter; T7A1, the T7A1/O4 promoter; tac, the tac promoter; lacUV5, the lacUV5 promoter; lac, the lac promoter; lacL8, the lacL8 promoter. (C) The percentage of hypernegatively supercoiled DNA for pZXD57 is a function of IPTG induction time. These results were calculated as described under Experimental Procedures using the TCDS data shown in (A). (D) The percentage of hypernegatively supercoiled DNA is proportional to promoter strength (the values are the average of at least three independent determinations and the standard deviations are shown). These results were calculated as described under Experimental Procedures using the TCDS data shown in (B). The promoter strength in P_bla units was obtained from ref. (Lanzer and Bujard 1988).

Figure 5. TCDS in E. coli topA strain VS111 for different plasmid DNA templates with identical size carrying a strong, IPTG-inducible P_T7A1/O4 promoter

The in vivo T-S assays were performed as described under Experimental Procedures. Transcription was induced with 1 mM of IPTG. Lane 1 contained the DNA sample before IPTG induction. Lanes 2–4 contained the plasmid DNA samples after 5, 10, and 15 min of IPTG induction, respectively. DNA topoisomers were resolved by electrophoresis in a 1% agarose gel containing 2.5 µg/ml chloroquine and stained with ethidium bromide. Plasmids pZXD60 (A), 62 (B), and 63 (C) carry a lacZ, GFPuv, and tet gene under the control of the strong P_T7A1/O4 promoter, respectively. (D) Plasmid pZXD60A is identical with pZXD60 except the start codon (ATG) of lacZ was mutated to the stop codon TAG (amber mutation). (E) Plasmid pZXD61 contains a lacZ gene in the reverse orientation, which cannot direct the expression of β-galactosidase. (F) Plasmid pZXD63A is similar to pZXD63 except the start codon of the tet gene was mutated to the stop codon TAG (amber mutation).

Figure 6. RT-PCR analyses of cDNA products of mRNA transcribed from different supercoiling-reporter plasmids pZXD60, 60A, 61, 62, 63 and 63A in E. coli topA strain VS111 harboring the linear plasmid pZXD51 after 10 min of IPTG induction

(A) RT-PCR experiments were performed as described under Experimental Procedures. The lower panel is a 1.2% agarose gel containing 1% formaldehyde to show the integrity of the RNA samples used for the RT-PCR experiments. The middle panel is a 12% polyacrylamide gel in 1×TAE buffer to show the PCR products of the cDNA synthesized from 16S rRNA samples isolated from E. coli strain VS111 carrying different supercoiling-reporter plasmids pZXD60, 60A, 62, 63, 63A, 57, and 59 after 10 min of IPTG induction. The upper panel is also a 12% polyacrylamide gel in 1×TAE buffer to show the PCR products of the cDNA samples synthesized from the mRNA samples isolated from E. coli strain VS111 carrying different supercoiling-reporter plasmids pZXD60 (lanes 1 and 4), 60A (lane 5), 62 (lane 2), 63 (lanes 3 and 6), 63A (lane 7), 57 (lane 8), and 59 (lane 9) after 10 min of IPTG induction. (B) Real-time RT-PCR analyses of the mRNA samples for E. coli strain VS111 carrying different supercoiling-reporter plasmids pZXD60, 60A, 61, 62, 63, 63A, and 59 after 10 min of IPTG induction (mean±SD, three independent experiments). Labels: lacZ, the lacZ gene; lacZA, the lacZ gene with an amber mutation in the start codon; lacZR, the lacZ gene in the reverse orientation; GFPuv, the GFPuv gene; GFPuvR, the GFPuv gene in the reverse orientation; tet, the tet gene; tetA, the tet gene with an amber mutation in the start codon.