Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins

Abstract

Transcription by RNA polymerase can stimulate localized DNA supercoiling in Escherichia coli. In vivo, there is extensive experimental support for a ''twin-domain'' model in which positive DNA supercoils are generated ahead of a translocating RNA polymerase complex and negative supercoils are formed behind it. Negative supercoils accumulate in the template DNA because the positive supercoils are preferentially removed by cellular topoisomerase action. Yet, in vitro, clear and convincing support for the twindomain mechanism has been lacking. In this article, we reconcile this inconsistency by showing that, in a defined in vitro system with plasmid DNA templates, a variety of sequence-specific DNAbinding proteins, such as the bacteriophage O replication initiator or the E. coli lactose or galactose repressors, strikingly stimulate transcription-coupled DNA supercoiling. We demonstrate further that this stimulation requires the presence in the DNA template of a recognition sequence for the relevant DNA-binding protein and depends on the production of long RNA chains by an RNA polymerase. Our data are most consistent with a model in which specific DNA-binding proteins facilitate a twin-domain mechanism to enhance DNA supercoiling during transcription. More precisely, we suggest that some nucleoprotein complexes, perhaps those that contain sharply bent DNA, can form barriers that impede the diffusion and merger of independent chromosomal supercoil domains. Localization of DNA supercoils by nucleoprotein complexes may serve as a general mechanism for modulating DNA transactions that are sensitive to DNA superhelicity.A number of DNA transactions in Escherichia coli are greatly facilitated by negative supercoiling of the bacterial genome (1). For example, opening of the DNA duplex during initiation of transcription at certain promoters and during initiation of DNA replication at the chromosomal origin is energetically favored when the DNA template is negatively supercoiled (see refs. 1 and 2 for reviews). The global DNA supercoiling level in E. coli is principally set through the opposing actions of DNA gyrase and DNA topoisomerase I (Topo I) (3, 4), although recent findings indicate that DNA Topo IV also participates in the relaxation of negatively supercoiled DNA (5).Several studies have demonstrated that the process of transcription of cellular DNA can enhance negative DNA supercoiling locally (3, 6-8). Liu and Wang formulated the ''twinsupercoiled-domain'' model (9) to explain this effect of transcription on DNA supercoiling. Extensive experimental support has been obtained in vivo (reviewed in refs. 1, 10, and 11) for the idea that positive DNA supercoils are generated in front of an advancing transcription complex while an equivalent number of negative DNA supercoils are formed behind it. Formation of these twin supercoiled domains occurs only when the translocating RNA polymerase and associated macromolecules are unable to rotate freely about the DNA helical axis. Under this condition, the template DNA rotates i...

Large-Scale Conformational Transitions in Supercoiled DNA Revealed by Coarse-Grained Simulation

“…The transition is marked by a progressive collapse in the radius of gyration with increasing jsj above jsj z 0.08, during which branched hyper-supercoils become increasing prominent in the heterogeneous conformational population. This final supercoiling regime coincides with the maximum degree of supercoiling that is accessible by DNA gyrase (28) and occurs within the range of supercoiling densities generated upstream of translocating RNA polymerase in in vitro and in vivo transcription experiments (29,30,58). The R G distributions for the 10-kilobase dataset in Fig.…”

“…This rotation generates positively supercoiled DNA in front of the translocating transcription machinery, and leaves hypernegatively supercoiled DNA in its wake. Consistent with this model, both in vitro and in vivo studies indicate that RNA polymerase is a powerful torque generator, and linking deficits below s ¼ À0.10 have been measured as a result of transcription at strong promoters both in the presence of functional Topo I (DNA relaxing enzyme) as well as in Topo I-deficient mutants (28)(29)(30). Studies in E. coli and Salmonella have shown that RNA polymerase is capable of actively creating and remodeling dynamic supercoiled topological domains (6,33).…”

“…The dependence of R H and other structural features on supercoiling density has been considered in detail in previous theoretical and simulation work (11,(13)(14)(15)(16)(17)(18). However, previous simulation work on plasmids with lengths exceeding six kilobases has been restricted to supercoiling densities up to s ¼ À0.06, which is only one-half the supercoiling density that is accessible to DNA gyrase (28) and well below the supercoiling density accessible to RNA polymerase (29,30). Although recent simulation work was conducted for supercoiled DNA rings with jsj as large as 0.086, this work was limited to plasmid sizes up to six kilobases (17), which is below the length scale where we observe the extended plectoneme to branched hyper-supercoil transition to occur.…”

“…However, the topological state of the bacterial chromosome is highly dynamic (27), and both DNA gyrase and RNA polymerase are capable of generating negative supercoiling densities far in excess of average supercoiling levels (28)(29)(30).…”

“…However, these larger linking deficits represent an important biological regime, because DNA gyrase is capable of generating supercoiling densities up to s~À0.12, and RNA polymerase has been observed to generate similar or greater linking deficits (28)(29)(30). Although simulation work on the scale of topological domains of E. coli has been previously reported, these studies were limited to supercoiling densities with jsj < 0.07 (11).…”