RNA polymerase II transcription termination is mediated specifically by protein binding to a CCAAT box sequence.

Abstract

A region in the adenovirus major late promoter (MLP) containing a CCAAT consensus sequence can direct transcription termination of RNA polymerase II, The mechanisms responsible for transcription termination of mRNA-encoding genes in eucaryotic cells are poorly understood. Several recent studies have shown that efficient termination of transcription by RNA polymerase II (pol II) requires a functional poly(A) addition site (8,22,25,36). However, transcription continues well beyond this site before terminating (for reviews, see references 4 and 31), and another signal that delineates the region or site at which termination occurs is necessary to direct efficient termination (9, 25). We have shown that a sequence containing an inverted CCAAT box consensus in the adenovirus major late promoter (MLP) functions as such a termination site signal (9). Since a number of proteins that bind CCAAT box sequences have been recently identified (6,7,10,20,26,32) and, more specifically, a protein purified from HeLa cells, CP1, has been shown to bind to the CCAAT sequence within the MLP (20), it is possible that protein binding to the MLP CCAAT box mediates termination induced by this sequence. However, the terminator regions analyzed previously all contained MLP sequences in addition to the CCAAT consensus. Furthermore, the ability of the fragments to induce termination was orientation dependent, perhaps suggesting that a function other than protein binding is involved.To gain insights into the mechanism by which this termination site functions, we have investigated the ability of a short oligonucleotide containing either the CCAAT consensus or mutant derivatives to elicit termination when inserted downstream of a poly(A) site. For these studies, p4)4-SVA, a chimeric plasmid that consists of the MLP (Ad2 nucleotides [nt] -405 to +33) directing transcription of the simian virus 40 (SV40) early region (SV40 nt 5171-2533) in a pBR322 vector (Fig. 1A) (24), was used to direct transcription in transient expression assays. The plasmid pSV2.CAT (12) was cotransfected in all cases to standardize transfection efficiencies (8, 9). Assays were performed using human 293 cells (1, 13), which have been shown to support high levels of transcription from p404-SVA (24 from -125 to -73 relative to the transcription start sites, and the other containing sequences from -85 to -51, were both capable of inducing termination when inserted into the SalI site (pBR322 nt 652) of p4)4-SVA located 330 base pairs (bp) downstream from the SV40 poly(A) site (9). These results together suggested that MLP sequences between -85 and -73 may be sufficient to induce termination.To test this hypothesis directly, a 13-bp oligonucleotide containing this sequence was inserted into the Sall site of p4)4-SVA (Fig. 1B). Plasmids containing single insertions in either the orientation in which this sequence is naturally found within the MLP, wt(1+), or in the opposite orientation, wt(1-), were identified and transfected into 293 cells. Forty-eight hours later, nuclei were ...

The conserved AAUAAA hexamer of the poly(A) signal can act alone to trigger a stable decrease in RNA polymerase II transcription velocity

“…Inserts iii and iv are G/U-less poly(A) signals. Insert iii has two hexamers and was included because some poly(A) signals contain two closely spaced hexamers (Connelly and Manley 1988;Natalizio et al 2002). Insert iv contains a single hexamer and is identical to insert iii except that the upstream hexamer in insert iii was mutated.…”

“…At the same time, certain mutants in yeast that cannot cleave at the poly(A) site nevertheless undergo poly(A)-dependent termination (Sadowski et al 2003;Zhelkovsky et al 2006), suggesting that poly(A) site cleavage may not be a fundamental part of the poly(A)-dependent termination mechanism. In another example, the poly(A) signal in mammals can alone drive termination in a heterologous sequence background (Orozco et al 2002), yet in their native context, some poly(A) signals require auxiliary elements to effect termination (Connelly and Manley 1989;Dye and Proudfoot 2001;West et al 2006).…”

Genes involved in pre-mRNA 3′-end formation and transcription termination revealed by a lin-15 operon Muv suppressor screen

“…When cleavage occurs at the poly(A) polymerase site, Pol II continues to transcribe a now-uncapped downstream RNA. This RNA is subject to degradation by the 5Ј-3Ј exonuclease XRN-2; according to the torpedo model, termination occurs when the exonuclease collides with Pol II (11)(12)(13). Recently, a unified allosteric-torpedo model has been proposed to explain new experimental evidence in support of both the earlier models (14,15).…”

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

“…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.…”