Transcription termination in animal viruses and cells

Resnekov, Orna; Ben-Asher, Edna; Bengal, Eyal; Choder, Mordechai; Hay, Nissim; Kessler, Mark A.; Ragimov, N; Seiberg, Miri; Skolnik-David, Hagit; Aloni, Yosef

doi:10.1016/0378-1119(88)90130-8

Cited by 29 publications

(25 citation statements)

References 31 publications

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“…Indeed, Aloni and colleagues have proposed that RNA secondary structure is important to the mechanism of pausing or termination at the ML site and other viral sites they have studied (reviewed in reference 1). However, our finding that base pairs upstream of +133 were not required for termination in vitro rules out the involvement of several specific potential secondary structures that Aloni and colleagues (24,39) have proposed to be important to the termination mechanism. If an RNA structure is required for pol II termination at the ML site, then either the RNA bases involved must lie entirely within the 50 bases upstream of the 3' end of the RNA or alternative structures involving sequences within the vector DNA must be contributing to termination.…”

Section: Resultssupporting

confidence: 45%

In vitro analysis of a transcription termination site for RNA polymerase II.

Wiest¹,

Hawley²

1990

Mol. Cell. Biol.

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Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

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Section: Resultssupporting

confidence: 45%

In vitro analysis of a transcription termination site for RNA polymerase II.

Wiest¹,

Hawley²

1990

Mol. Cell. Biol.

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“…Nascent RNA hairpins also are components of some eukaryotic regulatory mechanisms (e.g., HIV TAR RNA; ref. 10) and can trigger pausing or termination by RNA polymerase II in vitro (11). Although RNA hairpin-RNA polymerase interactions have long been suggested to play roles in pausing (12), termination (13)(14)(15), and antitermination (16), no direct evidence for these interactions or their effects exists.…”

mentioning

confidence: 99%

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

Wang¹,

Severinov²,

Landick³

1997

Proc. Natl. Acad. Sci. U.S.A.

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RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all -independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3 end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to ␤904-950 replaced crosslinking to ␤ and to other parts of ␤ that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3 end in the paused complex and be related to events at -independent terminators.The his leader pause hairpin is a well characterized nascent RNA structure that modulates RNA chain elongation (1-4). It forms concomitantly with a paused conformation of the transcription complex midway through the his biosynthetic operon leader region, where it increases the lifetime of the paused complex at least 6-fold. The pause allows time for a ribosome to initiate synthesis of the leader peptide and release the paused polymerase (probably by disrupting the pause hairpin), thereby synchronizing ribosome and polymerase movement during transcriptional attenuation (5). This and other pause sites are regulatory timing mechanisms that both prevent transcription beyond a region where regulatory input is effective and put polymerase in the proper conformation to interact with regulatory molecules.Similar hairpins are involved in some, but not all, pause signals; are required at -independent terminators (6-8), where they trigger dissociation of the transcription complex; and alone (e.g., HK022 put; ref. 9) or in association with proteins (e.g., N-nut complex; ref. 8) can function as antiterminators, which modify the transcription complex to block recognition of both pause sites and terminators. Nascent RNA hairpins also are components of some eukaryotic regulatory mechanisms (e.g., HIV TAR RNA; ref. 10) and can trigger pausing or termination by RNA polymerase II in vitro (11). Although RNA hairpin-RNA polymerase interactions have long been suggested to play roles in pausing (12), termination (13-15), and antitermination (16), no direct evidence for these interactions or their effects exists.The his pause RNA hairpin offers an excellent paradigm to study these interactions. It is one of four components of the multipartite his pause signal that also depends on a 3Ј-proximal region of RNA or DNA, alignment of the 3Ј-terminal nucleotide and the incoming NTP, and duplex DNA that contacts polymerase downstream from the active site (1, 2). Both the his pause signal and -independent terminators ...

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“…The three proto-oncogenes c-myc (52, 61, 65, 79, 85), c-myb (4, 62), and c-fos (16, 70) have been shown to be controlled at elongation. Adenovirus (37,66,73), simian virus 40 (36, 67), minute virus of mice (39), and human immunodeficiency virus (HIV) (40,41,75,83) have been demonstrated to have specific blocks to transcription elongation. The mRNA levels for the adenosine deaminase genes of humans and mice are at least partly controlled by a regulated block to elongation (13,14,42,48,58).…”

mentioning

confidence: 99%

Control of formation of two distinct classes of RNA polymerase II elongation complexes.

1992

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We have examined elongation by RNA polymerase II initiated at a promoter and have identified two classes of elongation complexes. Following initiation at a promoter, all polymerase molecules enter an abortive mode of elongation. Abortive elongation is characterized by the rapid generation of short transcripts due to pausing of the polymerase followed by termination of transcription. Termination of the early elongation complexes can be suppressed by the addition of 250 mM KCI or 1 mg of heparin per ml soon after initiation. Elongation complexes of the second class carry out productive elongation in which long transcripts can be synthesized. Productive elongation complexes are derived from early paused elongation complexes by the action of a factor which we call P-TEF (positive transcription elongation factor). P-TEF is inhibited by 5,6-dichloro-1-B-Dribofuranosylbenzimidazole at concentrations which have no effect on the initiation of transcription. By using templates immobilized on paramagnetic particles, we show that isolated preinitiation complexes lack P-TEF and give rise to transcription complexes which can carry out only abortive elongation. The ability to carry out productive elongation can be restored to isolated transcription complexes by the addition of P-TEF after initiation. A model is presented which describes the role of elongation factors in the formation and maintenance of elongation complexes. The model is consistent with the available in vivo data concerning control of elongation and is used to predict the outcome of other potential in vitro and in vivo experiments.It is now clear that the transcription of eucaryotic genes is controlled during the elongation phase as well as at initiation. The number of genes for which elongational control has been implicated is growing (80). The three proto-oncogenes c-myc (52, 61, 65, 79, 85), c-myb (4, 62), and c-fos (16, 70) have been shown to be controlled at elongation. Adenovirus (37,66,73), simian virus 40 (36, 67), minute virus of mice (39), and human immunodeficiency virus (HIV) (40,41,75,83) have been demonstrated to have specific blocks to transcription elongation. The mRNA levels for the adenosine deaminase genes of humans and mice are at least partly controlled by a regulated block to elongation (13,14,42,48,58). Many genes in Drosophila melanogaster have RNA polymerase II molecules arrested early after initiation (68,69). While control of elongation has been implicated in these examples, very little is known about the molecular mechanisms involved.A number of factors that influence elongation and termination by procaryotic RNA polymerase have been defined (86). In particular, the N and Q protein-mediated antitermination systems of lambda and similar bacteriophages provide a model for how specific gene expression can be controlled by modifying RNA polymerase elongation. The mechanism by which lambda Q protein functions has been partially elucidated (87). A key feature in this process is the pausing of the RNA polymerase downstream of the initiation...

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Transcription termination in animal viruses and cells

Cited by 29 publications

References 31 publications

In vitro analysis of a transcription termination site for RNA polymerase II.

In vitro analysis of a transcription termination site for RNA polymerase II.

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

Control of formation of two distinct classes of RNA polymerase II elongation complexes.

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