In prokaryotes, the response to thermal shock depends on temperature-regulated sigma factors. Concerted torpedo model for RNA termination of pole II. (A) Immediately after passing through the poly(A) site, the EC Pol II interrupts the transcription induced by one of the many mechanisms (see text); for example, by a conformational change including the finishing factors 3′ (green). (B) Endonucleolytic cleavage at the polyadenylation site and subsequent degradation of 3′ cleaved RNA by Rat1/Xrn2. At the same time, Rat1/Xrn2 recruits termination factors (term F) and transmits them to the active Pol II center. (C) The resulting Pole II is covered by the term F. RNA-binding proteins, in particular helicase-like ATPases such as TTF2 (see text), are candidates for the term F. It has been known for some time that the termination is functionally associated with cleavage and polyadenylation of the 3` end of the resulting transcription. In the two-step pre-m3` mRNA processing reaction, the transcription of the poly(A) signal triggers the endonucleolytic cleavage of the resulting transcript and produces an upstream fission product that is immediately polyadenylated (for examinations, see Colgan and Manley 1997; Zhao et al., 1999).
The fission product remaining downstream with an uncovered phosphate at its 5` end is highly unstable and degrades rapidly (e.B. Manley et al. 1982). In mammals, the majority of protein-coding genes contain a well-preserved hexanucleotide element (AAUAAA) positioned 10 to 30 nucleotides (nt) in front of the cleavage site, and a less conserved sequence rich in U or GU ∼10–30 nt downstream of the cleavage site. Multisubunit factors are responsible for the detection of these signals and the catalysis of fission and polyadenylation reactions. In mammals, these include the fission polyadenylation specificity factor CPSF; cleavage-stimulating factor, CstF; fission factors I and II, IPT and CFII; and poly(A) polymerase, PAP, which catalyzes the addition of adenosines to the poly(A) tail. Most components of these complexes have homologs in the yeast cleavage/polyadenylation apparatus, which consists of the cleavage polyadenylation factor, CPF, and the cleavage factors CFIA and CFIB. So we have an apparent “planning paradox.” The exonuclease activity of Rat1/Xrn2 appears to be essential for termination, but the resulting downstream RNA exonuclease degradation may not be.
One possibility is that exonuclease activity is necessary in some cases, but not in others. For example, if there is a strong poly(A) signal, an allosteric change is sufficient for the EC to enter a “time-ready” state. A strong poly(A) signal can contain pause-stimulating elements and associated factors. In all other cases, the timely attainment of this state would depend on the cotranscriptional degradation of the resulting transcription by Rat1/Xrn2 (Fig. 2). But then, how do we explain the EM data that most of the transcripts observed in fruit flies seem unlearned at the end (Osheim et al. 2002)? One possibility is that many poly(A) sites alone are sufficient to induce termination by allosteric modifications, thus avoiding the need for downstream RNA exonuclease degradation. However, the em results are not at odds with what we might call a “concerted torpedo model”, in which all the steps – fission/polyadenylation, exonucleolytic degradation and termination – are coupled into a single complex (Fig. 2). In fact, the finding that Rat1 appears to function as an integral part of the divided complex (Luo et al. 2006) supports this idea. Given that a rat1-associated protein, Rtt103, has been shown to be co-constructed with Pcf11 and other subunits of the CFIA cleavage/polyadenylation complex (Kim et al.
2004b), Luo et al. (2006) hypothesized that Rat1 could influence termination through interactions with the fission/polyadenylation apparatus. In support, the ChIP analysis showed that the recruitment of Rat1 at the 3` end of the ADH4 gene was reduced by the inactivation of Pcf11. Conversely, the inactivation of Rat1 reduced the networking of Pcf11 and Rna15 at the 3` end of the ADH4. These data suggest that Rat1 is necessary for optimal recruitment of cleavage/polyadenylation factors, which are also essential for discontinuation of treatment. To test whether Rat1 works in 3′ finish, they tested the effect of Rat1 on the choice of alternative poly(A) sites in act1 pre-mRNA. At the restrictive temperature, rat1-1 led to the preferred use of distal poly(A) sites at proximal sites, a characteristic of 3′ treatment factor mutants that inhibit cleavage/polyadenylation. However, the effect was significantly less severe than the poly(A) site displacement observed in strains with mutations in the CFIA`s Pcf11 subunit, and it is unclear whether this was a direct or indirect effect of rat inactivation1. Previous work has suggested, but has not shown, that Rat1 and Xrn2 degrade RNA by co-transcription (Kim et al., 2004b; West et al., 2004). To confirm this, Luo et al. (2006) used an RNA immunoprecipitation (RIP) analysis in which pole II is cross-linked to the resulting RNA downstream of the poly(A) site.
Detection of this RNA by RT-PCR after immunoprecipitation of Pol II indicates that it has not been degraded. Surprisingly, they found that inactivation of Rat1 alone did not result in downstream RNA accumulation. Only in rat1-1 xrn1Δ cells at the restrictive temperature was downstream RNA detectable, suggesting that Rat1 and Xrn1 may participate in cotranscriptional degradation. These data confirmed that downstream RNA degradation is cotranscriptional because in the presence of Rat1 or Xrn1 downstream RNA was not found to be associated with Pol II. Although Xrn1 is able to break down the nascent RNA downstream (at least in the absence of functional advice1), it does not work at the end of the phase (see above), suggesting that the resulting RNA degradation is not sufficient to cause termination. This is consistent with previous data showing that, in some situations, the termination may be defective, while cleavage and presumably exonuclease degradation of the downstream product are functional (Magrath and Hyman, 1999; Sadowski et al., 2003). Inhibition of the rho-dependent end by bicyclomycin is used to treat bacterial infections. The use of this mechanism with other classes of antibiotics is being studied to combat antibiotic resistance by removing protective factors in RNA transcription, while working synergistically with other gene expression inhibitors such as tetracycline or rifampicin.
[8] Although additional elements, such as breaks. B, contribute to the complexity of planning, the only sequence element typically required for termination is the poly(A) signal itself. In fact, poly(A) signal strength influences termination efficiency (Osheim et al., 1999; Orozco et al. 2002), suggesting that other elements play an alternative role. For example, they could become more important when effective and precise termination control is required, as in the case of the closely spaced adenovirus and complement genes mentioned above. In addition to the complexity of Pol II termination, it has also been found that the structure of chromatin throughout the terminating range affects the effectiveness of the termination. Cells lacking the chromatin remodeling factor Chd1 showed termination errors and defects in both chromatin structure at the terminating areas and at the termination itself (Alen et al. 2002). Chromatin remodeling at sites downstream of the Poly(A) site may be important to induce or facilitate the stopping or release of stencils by Pol II. It is clear that the presence of 3′ processing factors at the poly(A) signal and the resulting processes are essential for transcription termination, but what is the critical event that ensures that Pol II publishes the model? The importance of the exonucleases Rat1 and Xrn2 5′ → 3′ suggests that a conformational change in the EC, as proposed by the allosteric model, is not sufficient to promote termination and strongly supports the torpedo model.
On the other hand, the data discussed above, as well as recent evidence suggesting that downstream RNA exonuclease digestion is not enough to trigger termination, speak against the torpedo model in its simplest form. To remedy this, Luo et al. (2006) proposed a hybrid model in which Rat1 is an essential part of a Pol II complex that achieves cleavage at the poly(A) site, the resulting downstream RNA degradation, and the allosteric changes that promote its release from the model. But what these allosteric changes might be and how they would affect EC DISSOCIATION remains unclear. The best characterized termination factor is the bacterial protein ρ. ρ is a classical termination factor in that it provides a mechanism for dissociating emergent transcriptions in places where intrinsic terminators are missing. Rho is essential for the survival of most bacteria, although Rho is dispensable or completely absent in some prokaryotes. .