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Poly(A) signals and transcriptional pause sites combine to prevent interference between RNA polymerase II promoters.

Transcriptional termination by RNA polymerase II at the 3′ end of genes encoding poly(A)+ mRNAs is thought to require two distinct cis‐active elements: a functional poly(A) signal and a downstream transcriptional pause site. An important requirement for efficient termination is to prevent transcriptional interference of downstream‐located promoters. We have therefore investigated whether these two elements, individually or in combination, can prevent transcriptional interference of RNA polymerase II‐activated promoters. For this purpose, we constructed an expression plasmid containing two tandem retroviral long terminal repeats (LTRs) derived from HIV‐1. When transfected into HeLa cells, this construct resulted in transcriptional interference of the LTR promoters. Using this assay, we were able to show that a single poly(A) signal was able to protect an otherwise occluded promoter. This effect depended on the RNA‐processing strength of the poly(A) signal. Furthermore, transcriptional pause sites provided adequate protection against promoter occlusion even when tested alone. Finally, a combined element consisting of a poly(A) signal followed by a pause site was more efficient in promoter protection than either element on its own. These results indicate that an interference‐blocking element can take various forms: a poly(A) signal, a transcriptional pause site or a combination of both.