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.

Figure 1.

Transcription analyses of the lys2-CYCt-lacZ and lys2-lacZ fusion constructs. (A) Northern analyses of lys2-lacZ-containing mRNAs driven from the tet and GAL1 promoters. The 3-kb XbaI-EcoRV lacZ and a 589-bp 25 S rDNA internal fragments obtained by PCR (rRNA) were used as DNA probes. (B) Northern analyses of lys2-lacZ. The oligos C and W were used as single-stranded DNA probes. (C), (D) Northern analyses of pTGlylaT-1 and pTGlylaT-5 plasmids. Arrows refers to the mRNA being produced from the specified promoter. No arrow indicates that there is no transcription from that promoter. All experiments are made in the absence of doxycycline, so that the tet promoter is permanently active. A total of 2% glucose (Glu) or galactose (Gal) are used to either repress or activate the GAL1 promoter. Other details as described in ‘Materials and Methods’ section.

Figure 2.

Distribution of RNAPII along the lys2-lacZ transcription units of plasmids pTGlyla and pTGlylaTER. ChIP analyses in the wild-type strain (HRN1-4A) carrying the plasmids pTGlylaTER (A) or TGlyla (B). Schemes of the gene and the PCR-amplified fragments are shown. DNA ratios in regions 1–4 were calculated from the amounts obtained for these regions relative to the amounts of the intergenic region. ChIPs were performed from three independent cultures, and quantitative PCRs were repeated three times for each culture. SDs are indicated as error bars.

Figure 3.

Transcription analyses of lys2-CYCt-lacZ and lys2-lacZ fusion transcript driven form the GAL1 promoter. (A) Northern analyses of lys2-CYCt-lacZ containing mRNAs driven from the GAL1 promoter in wild-type (HRN1-4A), top1Δ (TOHR-11A), top2-1 (TORH-6A) and top1Δ top2-1 (TOHR-13C) strains. Mid-log phase plasmid-transformed cells were diluted in 3% glycerol–2%lactate synthetic complete (SC)-Trp medium plus doxycycline and diluted into identical fresh media to an OD₆₀₀ of 0.4 and incubated for 16 h. Galactose was then added and samples were taken for northern analyses at different times. RNA levels in arbitrary units were obtained by quantification of signals intensities in a FUJI FLA 5000 and normalized with respect to rRNA levels of each sample. Wild-type mRNA levels were taken as 100%. (B) Northern analyses of lys-lacZ-containing mRNAs driven from the GAL1 promoter using the same strains as in A.

Figure 4.

Northern analyses of convergent transcription in the lys2-CYCt-lacZ and lys2-lacZ fusion constructs. (A) Northern analyses of lys2-CYCt-lacZ mRNAs driven from the tet and GAL1 promoters in wild-type (HRN1-4A), top1Δ (TOHR-11A), top2-1 (TORH-6A) and top1Δ top2-1 (TOHR-13C) strains. The 3-Kb XbaI-EcoRV lacZ internal fragment were used as DNA probe. (B) Northern analyses of lys2-lacZ mRNAs driven from the P_tet and P_GAL1 promoters. Other details as described for Figure 3.

Figure 5.

Analysis of the effect of top1Δ and top2-1 on DNA supercoiling. (A) Chloroquine gel electrophoresis analysis of plasmid pTGlylaTER in wild-type (HRN1-4A), top1Δ (TOHR-11A), top2-1 (TORH-6A) and top1Δ top2-1 (TOHR-13C). Strains were cultured under non-transcription conditions in glucose plus doxycycline (Glu +dox) and under transcription conditions in galactose without doxycycline (Gal) at 30°C. Electrophoresis was carried out in the presence of 4 µg/ml chloroquine, and hybridization was performed with a labelled LYS2 DNA probe. Southern reveals bands representing topoisomers differing in linking number by steps of one. With the chloroquine concentration used, all topoisomers are negatively supercoiled, with those of increasing negative superhelicity migrating faster in the gel. The band marked as SC consists of the most negatively supercoiled species that are not resolved by the concentration of chloroquine used here. (B) Chloroquine gel electrophoresis analysis of plasmid pTGlyla.

Figure 6.

Effect of top1 and top2 mutations on transcription of lys-CYCt-lacZ and lys-lacZ fusion constructs at 37°C. (A) Northern analyses of lys-CYCt-lacZ-containing mRNAs driven from the tet and GAL1 promoters in wild-type (HRN1-4A), top1Δ (TOHR-11A), top2-1 (TORH-6A) and top1Δ top2-1 (TOHR-13C) strains at 37°C. Mid-log phase plasmid-transformed cells were diluted to an OD₆₀₀ of 0.4 synthetic complete (SGal)-Trp medium at 26°C and shifted to 37°C, samples were taken for northern analyses at different times. (B) Northern analyses of lys-lacZ mRNAs driven from the P_tet and P_GAL1 promoters. Other details as described in Figure 3.

Figure 7.

Recombination analyses of wild-type, top1Δ, top2-1 and top1Δ top2-1 strains. (A) Recombination between inverted repeat in plasmid pRS316-TINV carrying two inverted leu2 sequences. One copy is the leu2-HOr allele under the tet promoter and the other is a 5′-end truncated allele (leu2Δ5′). Transcription of leu2 is driven from tet promoter. Leu⁺ recombinants can arise by gene conversion of leu2-HOr without an associated inversion or by crossover occurring upstream of the HO site, whether or not associated with gene conversion. (B) Recombination between directs repeats in the system GL::PHO5 carrying a 600-bp truncated leu2 repeat under the P_GAL1 promoter. Leu⁺ recombinants can arise by the deletion of the PHO5 sequence. (C) Recombination between directs repeats in the system GL::LacZ carrying 600-bp truncated leu2 repeats under the P_GAL1 promoter. Leu⁺ recombinants arise by the deletion of the LacZ sequence.

Figure 8.

Recombination analyses of wild-type, top1Δ, top2-1 and top1Δ top2-1 strains in DNA substrates undergoing converging transcription. (A) Recombination frequencies were determined in wild-type (HRN1-4A), top1Δ (TOHR-11A), top2-1 (TORH-6A) and top1Δ top2-1 (TOHR-13C) strains transformed with plasmid pLlyla carrying the direct-repeat system L-Llyla in which transcription is under the P_LEU2 promoter. (B) Recombination frequencies in wild-type (HRN1-4A), top1Δ (TOHR-11A), top2-1 (TORH-6A) and top1Δ top2-1 (TOHR-13C) strains transformed with plasmid pLlylaTER carrying the direct-repeat system L-LlylaTER. For more details see ‘Materials and Methods’ section.