Poly(A)-Driven and Poly(A)-Assisted Termination: Two Different Modes of Poly(A)-Dependent Transcription Termination

Abstract

We mapped the elements that mediate termination of transcription downstream of the chicken betaH- and betaA-globin gene poly(A) sites. We found no unique element and no segment of 3'-flanking DNA to be significantly more effective than any other. When we replaced the native 3'-flanking DNA with bacterial DNA, it too supported transcription termination. Termination in the bacterial DNA depended on a functional poly(A) signal, which apparently compelled termination to occur in the downstream DNA with little regard for its sequence. We also studied premature termination by poorly processive polymerases close to the promoter. The rate of premature termination varied for different DNA sequences. However, the efficiencies of poly(A)-driven termination and promoter-proximal premature termination varied similarly on different DNAs, suggesting that poly(A)-driven termination functions by returning the transcription complex to a form which resembles a prior state of low processivity. The poly(A)-driven termination described here differs dramatically from the poly(A)-assisted termination previously described for the simian virus 40 (SV40) early transcription unit. In the SV40 early transcription unit, essentially no termination occurs downstream of the poly(A) site unless a special termination element is present. The difference between the betaH-globin and SV40 modes of termination is governed by sequences in the upstream DNA. For maximum efficiency, the betaH-globin poly(A) signal required the assistance of upstream enhancing sequences. Moreover, the SV40 early poly(A) signal also drove termination in betaH-globin style when it was placed in a betaH-globin sequence context. These studies were facilitated by a rapid, improved method of run-on transcription analysis, based on the use of a vector containing two G-free cassettes.

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

“…Using the reporter series shown in Figure 2A, in which the downstream cassette was placed at increasingly greater distances from the poly(A) signal, Orozco et al (2002) detected an increase in polymerase density in the downstream cassette (pausing) when it was close to the poly(A) signal, but exponentially decreasing polymerase densities (termination) as the cassette was placed increasingly farther away. Results obtained by using cassettes (Yeung et al 1998;Steinmetz and Brow 2003;Zhelkovsky et al 2006) are comparable to those obtained by slot blot hybridization FIGURE 2. The G/U-rich region of the poly(A) signal is not required for pausing.…”

“…In both cases only T1 RNase was used. (Yeung et al 1998;Dichtl et al 2004), but cassettes offer various theoretical and practical advantages, as discussed previously (Yeung et al 1998;Orozco et al 2002). Figure 1B shows the cloning inserts that we have used to evaluate the role of the G/U-rich region.…”

Assembly of the Cleavage and Polyadenylation Apparatus Requires About 10 Seconds In Vivo and Is Faster for Strong than for Weak Poly(A) Sites

“…8B results from events taking place at the SV40 early poly(A) site. Although it is formally possible that transcription termination between sense and antisense sequences contributes to the rescue, this is not likely because poly(A) sites do not drive termination in the plasmid background we have used (67). We should also point out that although the 1,387-nt antisense sequence happens to contain the SV40 late poly(A) site (10), the presence of the complementary sense sequence upstream ensures that this downstream site never becomes accessible for processing (Fig.…”

“…Transfections for chloramphenicol acetyltransferase (CAT) assays were performed by the calcium phosphate method as described previously (67). The transfected DNA included a cotransfected luciferase-expressing plasmid, pRSVfl, in which the HindIII-SacI fragment of Promega's pGem-luc replaces the HindIII-BalI segment of pRSVcat.…”

Poly(A) signal-dependent degradation of unprocessed nascent transcripts accompanies poly(A) signal-dependent transcriptional pausing in vitro

“…Poly(A) signal-dependent termination can occur independently of sequence downstream from the poly(A) signal (Yeung et al 1998). To establish whether poly(A) signaldependent pausing also is independent of downstream sequence, the various pausing experiments in Figures 1 and 3 were carried out using two different reporters having completely unrelated sequences between their cassettes.…”