Transcriptional collision between convergent genes in budding yeast

Abstract

Transcriptional interference between genes and the regulatory elements of simple eukaryotes such as Saccharomyces cerevisiae is an unavoidable consequence of their compressed genetic arrangement. We have shown previously that with the tandem arranged genes GAL10 and GAL7, inefficient transcriptional termination of the upstream gene inhibits initiation of transcription on the downstream gene. We now show that transcriptional interference can occur also with S. cerevisiae RNA polymerase II genes arranged convergently. We demonstrate that when the GAL10 and GAL7 genes are rearranged in a convergent orientation, transcriptional initiation occurs at full levels. However, as soon as the two transcripts begin to overlap, elongation is restricted, resulting in a severe reduction in steady-state mRNA accumulation. This effect is observed only in cis arrangement, arguing against RNA-interference effects acting on the potential generation of antisense transcripts. These data reinforce the necessity of separating adjacent RNA polymerase II transcription units by efficient termination signals.

Figure 1

TRO analysis of transformed GAL10-7 plasmids. (A) TRO analysis of pYC10-7 (WT) transformed cells. The diagram shows the tandem arrangement of the GAL10 and GAL7 genes, with the direction of transcription indicated with an arrow. The position of the single-stranded M13 probes used are shown and are as described in Table 1. Probe m is an M13 probe without an insert serving as a negative hybridization control. Probe a is an M13 probe with a fragment of the S. cerevisiae actin gene serving as a positive transcription control. The distances between the ORFs of GAL10 and GAL7 in the WT orientation are labeled. Open arrowheads indicate polyadenylation sites. The GAL7 promoter region is indicated also. (B) TRO analysis of pYC10-7Con (Con)-transformed cells. The diagram shows convergent orientation of GAL10 and GAL7, with the intergenic region's distance labeled. This construct includes 240 bp downstream of GAL7 and 293 bp downstream of GAL10 to make a hybrid intergenic region. Sense and antisense single-stranded M13 DNA probes were selected as appropriate. (C) Quantitation of the data shown in A and B. Signals were quantified in a PhosphorImager (Molecular Dynamics). The values were corrected for background hybridization and U content in each probe. Signals were normalized to the actin positive transcription probe a on each filter to allow direct comparison of transcription levels.

Figure 2

Steady-state analysis of GAL constructs. Northern blot with total RNA from pYC10-7 (WT, lane 1), pYC10-7Con (Con, lane 2), pYC10-7Fus (Fus, lane 3), Fus-Δ7P (lane 4), Fus-Δ10P (lane 5), Fus-Δ7P/Δ10P (lane 6), and Fus-Δ7P&Δ10P (lane 7). Longer exposure shows the read-around bands for Fus-Δ7P (lane 4) and Fus-Δ10P (lane 5). The filter was striped and reprobed with ACT1 as a loading control. Quantitated levels of steady-state GAL10 and GAL7 mRNA are corrected for actin and compared by using the steady-state level of GAL10 and GAL7 mRNA from WT as 1.

Figure 3

TRO analysis of transformed plasmids as indicated (A–D). The positions of single-stranded M13 probes used are shown in the diagram at the bottom of the figure and are as described in Table 1.

Figure 4

Quantitation of Fus and Fus-Δ7P and Fus-Δ10P transcription profiles. (A) TRO analysis of Fus transformed plasmid. The signals were corrected as described for Fig. 1. Arrows indicate the directions of transcribing polymerases. (B) TRO analysis of Fus-Δ7P and Fus-Δ10P. In the Fus-Δ10P plasmid (giving GAL7 transcription), the O probes have been deleted with the deletion of the GAL10 promoter. In the GAL10 transcription profile, decrease in GAL10 transcription over probe A is likely the result of cryptic polyadenylation that is seen at the steady-state level (Fig. 2, lane 4). Also, the above background levels of transcription over probe O (before the GAL10 promoter) are most likely to be the result of read-around transcription seen at steady state (Fig. 2, lane 4). The single-stranded M13 probes used are described in Table 1.