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.
Eggermont
J
J
Sir William Dunn School of Pathology, University of Oxford, UK.
Proudfoot
N J
NJ
D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D011061:Poly A / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000235:genetics*; D013728:Terminator Regions, Genetic
D011061:Poly A; D012319:RNA Polymerase II