A pause site for RNA polymerase II is associated with termination of transcription.

Abstract

Termination of transcription by RNA polymerase II has been postulated to involve a pausing process. We have identified such a pause signal, 350 bp into the 3′ flanking region of the human alpha 2 globin gene at a position where termination is thought to occur. We show that this pause signal enhances the utilization of an upstream poly(A) site which is otherwise out‐competed by a stronger downstream poly(A) site. We also demonstrate that the pause site rescues a poly(A) site that is inactive due to its location within an intron. Using nuclear run‐on analysis we show that elongating RNA polymerase II molecules accumulate over this pause signal. Furthermore we show that when the pause site is positioned immediately downstream of a strong poly(A) signal, significant levels of transcription termination take place.

Polyadenylation and transcription termination in gene constructs containing multiple tandem polyadenylation signals

“…Results from a number of laboratories have shown that when two polyadenylation sites are duplicated and separated by relatively short distances, the upstream polyadenylation site is used preferentially (19,28,(45)(46)(47)(48). However, one study showed that for duplicated poly(A) sites, a stronger downstream one could outcompete a weaker upstream one when less than 400 bp away (19).…”

“…However, one study showed that for duplicated poly(A) sites, a stronger downstream one could outcompete a weaker upstream one when less than 400 bp away (19). In those instances where RNA levels were measured, there was no significant change in mRNA amounts produced by constructs with multiple signals (28,45,47).…”

“…RNA polymerase II transcription termination occurs hundreds or thousands of nucleotides downstream of polyadenylation sites (7,13,14). Although there have been a number of reports of specific sequences which may act as termination sequences (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), in vivo, transcription termination often depends on 3'-end processing (9)(10)(11)(12)26). Mutations in the conserved polyadenylation AAUAAA signal of the mouse major 3-globin gene or the human globin gene, respectively (11,12), or mutations in both the AAUAAA hexanucleotide and the downstream GT-rich region of the SV40 early polyadenylation site (9) can inhibit polyadenylation as well as transcription termination.…”

Transcription termination by nuclear RNA polymerases

“…This increased RNAPII density at the 39-end may reflect pausing of RNAPII that precedes termination. Indeed, transcription pause sites located downstream from poly(A) sites appear to play a role in RNAPII 39-end processing and termination, at least in mammalian genes, such as a-globin and g-globin genes (Enriquez-Harris et al 1991;Plant et al 2005). A G-rich sequence (MAZ element: G 5 AG 5 ) was identified downstream from the poly(A) site of the liver-specific C2 complement gene (Ashfield et al 1994;Proudfoot 1999, 2000).…”

Intergenic transcription and transinduction of the human β-globin locus

“…As mature transcripts are generally mapped by cDNA cloning, the extent of most primary transcription units is unknown. Nuclear run-on (NRO) analysis, which measures polymerase density, has generally been applied only to single genes and their immediate flanking regions (Hagenbuchle et al 1984;Ashfield et al 1991;Enriquez-Harris et al 1991). Therefore, transcription of complete intergenic regions in gene loci has not been investigated.…”