Transcription and supercoiling of the template DNA are closely related each other. DNA supercoiling affects transcription and transcription affects supercoiling of the template DNA. Furthermore, packaging of genomic DNA into chromatin in eukaryotes raises another type of relation. DNA supercoiling can affect transcription through modulation of the chromatin structure.

Introduction

Typical double-stranded DNA consists of a helix with a pitch of one turn per 10.5 base pairs. Both underwinding and overwinding of the DNA double helix induce twisting and coiling of the helix unless the DNA strand can rotate freely. The coils thus formed are termed negative and positive supercoils, respectively (see Chapter 1 for details). Current views of the mechanisms underlying transcriptional regulation rely on a concept originally proposed by Jacob and Monod: regulation through cis-elements on DNA and trans-acting factors that bind to the elements.¹ However, DNA supercoiling can modulate accessibility of trans-acting factors to cis-elements. Transcription is an asymmetric process: only one strand of the DNA double helix is copied into RNA. To achieve this, the double helix must be locally unwound. Therefore, negative supercoiling of DNA is thought to facilitate the step. Moreover, DNA supercoiling can affect transcription in chromatin context in eukaryotes. This chapter discusses roles of DNA supercoiling in transcriptional regulation.

Prokaryotic Transcription

In prokaryotes, there are two major topoisomerases that act toward opposite direction. ² DNA gyrase can generate negative supercoils into relaxed DNA and relax positively supercoiled DNA. In contrast, topoisomerase I can relax negatively supercoiled DNA but not positively supercoiled DNA. The superhelical state of cellular DNA in prokaryotes appears to be under equilibrium between actions of these topoisomerases. Measurements of psoralen binding averaged globally across the Escherichia coli genome have detected unconstrained negative supercoils with a superhelical density of —0.05.³

Consistent with the finding of unconstrained negative supercoils in genomic DNA, it has been established that DNA supercoiling functions as a regulator of prokaryotic transcription.⁴ First, supercoiling affects transcription in vitro. Some promoters have an optimum level of supercoiling for their transcription and the level is different for different promoters. Second, supercoiling plays a regulatory role in vivo. Genetic studies have shown that mutations in topoisomerase I⁵ or DNA gyrase⁶ affect transcription in vivo.

Two rate limiting steps are known for prokaryotic transcription. One is formation of an RNA polymerase-DNA open complex and the other is promoter clearance. Because negative supercoiling favors the unwinding of the DNA double helix that is required for formation of the open complex, it is expected to increase the rate of transcription for promoters in which open complex formation is rate limiting. Indeed most genes are activated by increased negative supercoiling. However, transcription of gyr A and gyr B encoding the subunits of DNA gyrase is induced by relaxation of DNA. It has been proposed that promoter clearance but not open complex formation is the rate-limiting step for these promoters.⁷

The superhelical state of DNA is known to change depending on the growth conditions of cells.⁸ For example, nutrient downshift and stationary growth phase cause a decrease in the extent of negative supercoiling, while high osmolarity leads to an increase in the negative supercoiling of DNA. In agreement with these findings, transcription by RNA polymerase harboring the stationary phase-specific σ^s appears to be enhanced on templates with decreased superhelicity.⁹ DNA supercoiling also changes transiently during heat shock.¹⁰ The heat stress induces rapid relaxation of negative supercoils and then DNA topology returns back to the original state with negative supercoiling. In response to the relaxation, most genes are repressed but some specific or stress genes are induced.¹¹

Transcription of a double helical DNA requires a rotation of an RNA polymerase and its nascent RNA chain relative to DNA. The velocity of the rotation is calculated to be a few hundred rounds per minute since a pitch of the helix is about 10 base pairs and the rate of RNA chain elongation is a few thousand nucleotides per minute. It seems difficult for the RNA polymerase and its nascent RNA to rotate around the template DNA at such a high velocity. Instead the DNA must turn around its axis during transcription. Then the tracking RNA polymerase generates positive supercoils in the template DNA ahead of it and negative supercoils behind it (fig. 1). These supercoils will be relaxed by DNA topoisomerases. Liu and Wang summarized the concept as the twin-supercoiled-domain model.¹² The model predicts the followings. First, negative supercoils should accumulate in the absence of a negative supercoil-relaxing enzyme. Second, positive supercoils should accumulate in the absence of a positive supercoil-relaxing enzyme. These predictions are fulfilled by two earlier observations. First, pBR322 DNA harboring high degrees of negative supercoiling has been isolated from topA mutants of E. coli and Salmonella typhimurium, and the presence of the highly negatively supercoiled DNA was dependent on the transcription of the tetA gene.¹³ Second, highly positively supercoiled pBR322 DNA has been isolated from E. coli treated with gyrase inhibitors.¹⁴ According to the model, negative and positive supercoils would accumulate in the intergenic regions of two divergent and convergent transcription units, respectively. Such supercoils can in turn affect transcription.

Figure 1

Transcription-driven supercoils. Tracking RNA polymerase generates positive supercoils in the template DNA ahead of it and negative supercoils behind it. “B” represents a topological barrier. It does not necessarily require attachment (more...)

Eukaryotic Transcription

In eukaryotes, psoralen-binding assays on human HeLa and Drosophila Schneider cell lines have shown that the bulk of each genomic DNA is torsionally relaxed within nuclei.³ However, it did not necessarily exclude a possibility that there were negatively supercoiled micro domains within these genomes. Indeed unconstrained negative supercoils have been demonstrated in the hsp70 and 18S-ribosomal RNA genes of the Schneider cell line,¹⁵ and in the dihydrofolate reductase gene¹⁶ and the hygromycin resistance transgenes¹⁷ of cultured human cells.

To test whether the twin-supercoiled-domain model is applicable to eukaryotic transcription, Giaever and Wang constructed a yeast plasmid carrying the coding sequence of the E. coli topA gene placed downstream of an inducible yeast promoter.¹⁸ The plasmid DNA became positively supercoiled in yeast ΔtopI top2ts cells at the restrictive temperature when the E. coli DNA topoisomerase I was expressed. The generation of positive supercoils was observed only during transcription. Because neither one of the yeast DNA topoisomerases I and II can be functional under these conditions and because the E. coli enzyme can relax only negative supercoils, these results verify the model. Recently Matsumoto and Hirose¹⁹ have visualized transcription-coupled, unconstrained negative supercoils of DNA in approximately 150 loci on polytene chromosomes of Drosophila melanogaster. The results demonstrate that transcription-coupled negative supercoils of DNA exist within a cell even in the presence of active topoisomerases. These negative supercoils can affect transcription.

As described for prokaryotic transcription, supercoiling modulates in vitro transcription of eukaryotic genes.²⁰ Hirose and Suzuki,²¹ and Mizutani et al²² have shown that transcription of the Bombyx mori fibroin gene increases and plateaus from templates of increasing negative supercoiling, and transcription from the adenovirus type 2 major late promoter (Ad2MLP) rises and then falls, while transcription of the Drosophila hsp70 gene remains unchanged (fig. 2). Dissection of transcription revealed that formation of a preinitiation complex on the fibroin gene or the Ad2MLP is slow on relaxed DNA and accelerated by negative supercoiling of DNA. On the contrary, the preinitiation complex assembled rapidly on the hsp70 gene irrespective of DNA topology. Tabuchi et al23 have demonstrated that binding of TATA element binding protein (TBP) to the TATA element induces underwinding of the duplex DNA (approximately 0.5 linking difference per bound TBP molecule as shown in fig. 3). The underwinding has been confirmed by crystal structure of a TBP-TATA element complex.^24,25 The change was facilitated by negative supercoiling of DNA on the fibroin promoter and the Ad2MLP but not on the hsp70 promoter.²³ These data reveal that although supercoiling can affect both prokaryotic and eukaryotic transcription in vitro, the critical steps are different: open complex formation in most genes of prokaryotes vs TBP binding to the TATA element in most genes of eukaryotes. In transcription from the Ad2MLP, promoter clearance is also facilitated by negative supercoiling of DNA.²⁶ It is possible that clearance of the Ad2MLP goes up and then down with increasing negative supercoiling. Interestingly, the rate-limiting step in hsp70 transcription is not preinitiation complex formation but restart of a paused RNA polymerase to productive elongation.²⁷ Probably hsp70 transcription becomes independent of DNA topology so that it can be induced immediately upon heat shock.

Figure 2

DNA superhelicity affects transcription of eukaryotic genes differently. In vitro transcription activities of indicated promoters were measured on plasmid DNAs with various superhelical densities. Reprinted from Mizutani M, Ura K, Hirose S. Nucl Acids (more...)

Figure 3

Underwinding of DNA upon binding of TBP to the TATA element. Negatively supercoiled plasmid DNA carrying Ad2MLP was incubated with indicated concentration of yeast TBP at 30°C and then treated with DNA topoisomerase I. DNA was purified and analyzed (more...)

Whether DNA supercoiling affects transcription in vivo is still elusive in eukaryotes. Although Dunaway and Ostrander have clearly shown that local domains of negative supercoiling activate the ribosomal RNA gene promoter in vivo,²⁸ other promoters have not been tested through a similar approach. Supercoiling factor (SCF) is a protein capable of generating negative supercoils in relaxed DNA in conjunction with topoisomerase II.²⁹ D. melanogaster SCF localizes to many interbands and puffs that are active sites of transcription³⁰ (fig. 4), suggesting that SCF plays a role in formation of transcriptionally active chromatin. Recent study has shown that transcription of certain genes is compromised by targeting SCF with RNAi (Furuhashi and Hirose, unpublished). Because SWI/SNF-type chromatin remodeling factors require changes in DNA topology for their action,³¹ SCF and topoisomerase II may facilitate chromatin remodeling through generation of negative supercoils.

Figure 4

Drosophila SCF localizes to interbands and puffs on polytene chromosomes. Salivary gland polytene chromosomes were stained with antibody against SCF (red) and DAPI (blue). Reprinted from Kobayashi M, Aita N, Hayashi S et al. Mol Cell Biol 1998; 18:6373-6744 (more...)

Formation of unusual DNA structure (see Chapter 1 for details) such as Z-form is significantly facilitated by negative supercoiling.³² Such unusual DNA structure can affect transcription. For example, Z-DNA in a promoter region has been suggested to participate in transcriptional activation in collaboration with a chromatin remodeling complex BAF.³³ On the contrary, Z-form within a transcribable region would inhibit transcription elongation. Finally positive supercoiling of DNA has been reported to diminish transcription in vivo.³⁴ These data indicate a possible role of DNA supercoiling in eukaryotic transcription in vivo.

Conclusion

DNA supercoiling can affect transcription in both prokaryotes and eukaryotes. While the critical step is open complex formation in most prokaryotic genes, TBP binding to the TATA element is affected in most eukaryotic genes. Furthermore, DNA supercoiling can affect transcription through chromatin remodeling in eukaryotes.

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