Topological constraints impair RNA polymerase II transcription and causes instability of plasmid-borne convergent genes

Abstract

Despite the theoretical bases for the association of topoisomerases and supercoiling changes with transcription and replication, our knowledge of the impact of topological constraints on transcription and replication is incomplete. Although mutation of topoisomerases affects expression and stability of the rDNA region it is not clear whether the same is the case for RNAPII transcription and genome integrity in other regions. We developed new assays in which two convergent RNAPII-driven genes are transcribed simultaneously. Plasmid-based systems were constructed with and without a transcription terminator between the two convergent transcription units, so that the impact of transcription interference could also be evaluated. Using these assays we show that Topos I and II play roles in RNAPII transcription in vivo and reduce the stability of RNAPII-transcribed genes in Saccharomyces cerevisiae. Supercoiling accumulation in convergent transcription units impairs RNAPII transcription in top1Δ strains, but Topo II is also required for efficient transcription independent of Topo I and of detectable supercoiling accumulation. Our work shows that topological constraints negatively affect RNAPII transcription and genetic integrity, and provides an assay to study gene regulation by transcription interference.

DNA Topoisomerase Iα Affects the Floral Transition

“…These observations demonstrate a pivotal role of DNA topoisomerase I in transcriptional initiation. In contrast, some other studies have suggested that DNA topoisomerase I also affects transcriptional elongation by resolving unfavorable supercoils generated in the course of the movement of RNA polymerase II (Mondal et al, 2003;García-Rubio and Aguilera, 2012;King et al, 2013). Our results demonstrate that TOP1a facilitates the recruitment of RNA polymerase II to the regions near the transcriptional start sites of FLC, MAF4, and MAF5 loci but does not affect the occupancy of RNA polymerase II in the other regions, suggesting that Arabidopsis TOP1a specifically affects transcriptional initiation rather than transcriptional elongation at target genes during the floral transition.…”

Dissecting the Roles of Divergent and Convergent Transcription in Chromosome Instability

“…The events surrounding collision of RNA polymerases during transcription are complex and of universal relevance for all living organisms (Garcia-Rubio and Aguilera, 2012; Hobson et al, 2012; Liu and Alberts, 1995; Prescott and Proudfoot, 2002). Much of the early analysis of this phenomenon was from work performed in bacteria where it became clear that not only must the steric nature of two transcription complexes be considered, but also the effect that the complexes have on the DNA they are transcribing.…”

“…While convergently expressed genes would have their promoter regions separated by the two gene bodies, some convergent promoters may be close enough to occupy the same NDR. Work has shown that as two RNAP2 complexes approach each other from convergent promoters in a head-on collision, the two complexes stall and prevent each other from proceeding (Garcia-Rubio and Aguilera, 2012; Hobson et al, 2012; Saeki and Svejstrup, 2009). However, the consequences of very close convergent promoters in vivo has only recently begun to be studied, as it now appears that antisense transcription can lead to convergent transcription, especially at proposed super-enhancer regions (Lu et al, 2015; Meng et al, 2014).…”

RNA Polymerase Collision versus DNA Structural Distortion: Twists and Turns Can Cause Break Failure

“…Interestingly, we found that the collision of sense and antisense RNAP2 complexes emanating from convergent promoters did not increase GCRs as measured in the assay. While single subunit bacteriophage RNA polymerases are small enough to bypass each other when transcribing convergently (Ma and McAllister, 2009), both in vitro and in vivo assays suggest that when two eukaryotic RNAP2 complexes collide, each pauses until one or both are removed (Garcia-Rubio and Aguilera, 2012; Hobson et al, 2012). Therefore, physical strain or stalling of two converging complexes does not seem to present a danger for forming a GCR in wild-type cells; in contrast, the DNA structure left in the wake of two RNAP2 complexes traveling on the DNA in opposite directions does (Figure 1A), suggesting that the underwound DNA topology may be a key potentiator of DNA DSBs.…”

“…Convergent promoters have no effect in the assay and is likely explained by evidence that two RNAP2 complexes that collide head-on will stall until they are removed (Hobson et al, 2012). While this likely affects transcription of the genes in the vicinity of the stall site (Garcia-Rubio and Aguilera, 2012), recombination is not detected in wild-type cells to a significant degree.…”