In vitro formation of short RNA polymerase II transcripts that terminate within the HIV-1 and HIV-2 promoter-proximal downstream regions.

Toohey, M G; Jones, Katherine A.

doi:10.1101/gad.3.3.265

Cited by 122 publications

(95 citation statements)

References 45 publications

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“…The consistency in the lengths of these processed RNAs indicates that transcription preferentially started at the tailed junctions, as previously reported (26). To test that the processing activity was as expected (19,20), we added a pre-RNA (made by SP6 RNA polymerase) corresponding to the full-length transcript of poly(dC)-tailed template 2 and found that the products generated during ongoing transcription (lane 4) corresponded to those obtained [3][4][5] were also labeled by the CCA-end exchange activity in the extract (27). This, however, also served as an internal control for RNA recovery, and lane 6 shows that there are no other products obscured by the strong tRNA signal.…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Premature termination (attenuation) in the 5'-proximal regions of several genes in mammalian systems has been examined by either promoter-directed transcription in cell extract systems (4-10) or promoterless transcription with purified pol II (11-14). These sites may be intrinsic terminators for pol II and contain distinct signals such as RNA stem-loop structures (5,7,14), DNA bending in T-rich tracts (13), or other, structureless sequences (6).While these examples illustrate an important role for transcription attenuation in the regulation of eukaryotic gene expression, the fate of pol II in 3' noncoding regions after it passes the poly(A) site(s) and terminates at downstream sequences is obscure, both temporally and mechanistically (15). In higher eukaryotes, mutation studies and nuclear run-on experiments have suggested that the RNA processing events associated with mature 3'-end formation may themselves be involved in regulating the efficiency oftranscription termination, despite the progress of pol II for long distances past the poly(A) site (16-18).…”

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Transcriptional arrest of yeast RNA polymerase II by Escherichia coli rho protein in vitro.

Wu¹,

Platt²

1993

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

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A promoter-independent assay utilizing poly(dC)-tailed DNA templates has revealed that Saccharomyces cerevisiae whole-cell extracts can be proficient for transcription by the endogenous yeast RNA polymerase H as well as for correct 3'-end RNA processing. Our attempts to examine the fate of polymerase II itself were inconclusive, because only trace btanscription products corresponded to the expected size of terminated RNA species. Transcription in our processingproficient extract was thus insufficient to cause termination. To test our system with a known, albeit heterologous, signal, we examined a dC-tailed template carrying the E. col rhodependent termination signal £p t' in the yeast extract. Transcripts from this template were not susceptible to processing, but addition of rho protein resulted in two distinct truncated transcripts that could not be chased by excess unlabeled nucleotides. These RNA species thus represented stably paused or terminated polymerase II products, and their absence when a mutated unresponsive tip t' template was used affirmed that they were due to the effects of rho. E. col RNA polymerase added to a yeast extract pretreated with a-amanitin was also halted by rho at these same two sites. A mutated rho protein, while only partly defective with E. coli polymerase, failed to provoke arrest when transcription was carried out by RNA polymerase H. Thus, functional rho and its cognate site, tip t', appear necessary and sufficient to elicit the production of truncated transcripts by RNA polymerase II in a yeast wholecell extract. The ability of rho to halt the eukaryotic enzyme strengthens the likelihood that a rho-like helicase may be involved in RNA polymerase H transcription termination.Transcription termination is required to complete RNA synthesis and prevent transcriptional interference with downstream genes. It occurs when the RNA polymerase dissociates from the DNA template and the nascent RNA is released. Termination mechanisms are classified into two major categories (1). Intrinsic termination occurs as RNA polymerase stops in direct response to signals encoded in the RNA transcript and/or the DNA template. Alternative mechanisms require the action of some additional trans-acting factor(s) to terminate RNA elogation by the polymerase molecules. Among identified bacterial termination factors, the involvement of Escherichia coli rho protein in transcription termination is best understood (2).In eukaryotes, the study of RNA polymerase II (pol II) termination is complicated by 3'-end RNA processing, which produces mature mRNA 3' ends via specific endonucleolytic cleavage and polyadenylylation. To distinguish between termination and RNA processing events, nuclear run-on studies in vivo or transcription experiments in vitro are required. However, the former are tedious and time-consuming, and the latter are difficult due to the complexities of pol II The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adv...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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Transcriptional arrest of yeast RNA polymerase II by Escherichia coli rho protein in vitro.

Wu¹,

Platt²

1993

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

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“…4C) is based on the observation that prematurely terminated HIV RNAs are stable (31,34). The TM RNase protection bands represent a diffuse set of RNAs with 3Ј ends in the HIV-2 TAR sequence, which acts as a terminator and/or stabilizer of terminated transcripts (66). The RT RNase protection product represents transcripts which initiate correctly and extend beyond position ϩ165.…”

Section: To 4)mentioning

confidence: 99%

Three Functional Classes of Transcriptional Activation Domains

Blau

Xiao

McCracken

et al. 1996

Molecular and Cellular Biology

258

263

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We have studied the abilities of different transactivation domains to stimulate the initiation and elongation (postinitiation) steps of RNA polymerase II transcription in vivo. Nuclear run-on and RNase protection analyses revealed three classes of activation domains: Sp1 and CTF stimulated initiation (type I); human immunodeficiency virus type 1 Tat fused to a DNA binding domain stimulated predominantly elongation (type IIA); and VP16, p53, and E2F1 stimulated both initiation and elongation (type IIB). A quadruple point mutation of VP16 converted it from a type IIB to a type I activator. Type I and type IIA activators synergized with one another but not with type IIB activators. This observation implies that synergy can result from the concerted action of factors stimulating two different steps in transcription: initiation and elongation. The functional differences between activators may be explained by the different contacts they make with general transcription factors. In support of this idea, we found a correlation between the abilities of activators, including Tat, to stimulate elongation and their abilities to bind TFIIH.Stimulation of eukaryotic gene expression requires sequence-specific factors with DNA binding domains and activation domains that interact with the general transcription factors (GTFs) and recruit RNA polymerase II (pol II) to the promoter (3, 65). In vivo the transcriptionally active form of pol II is probably a holoenzyme complex which contains a number of the GTFs as well as other polypeptides (36). Different activation domains interact with different GTFs, including TFIIB (44), TFIID (17,19,63), and TFIIF (76). These interactions are thought to recruit, stabilize, and/or modify the activity of the pol II holoenzyme.We recently demonstrated that the activation domains of p53, VP16, and E2F1 bind directly to TFIIH (50a, 72). TFIIH is a multisubunit factor, different forms of which are required for both transcription and nucleotide excision repair of DNA (9). TFIIH is the only GTF which has enzymatic activities: it has two helicase subunits and a cyclin-dependent protein kinase subunit which phosphorylates the pol II large-subunit C-terminal domain (CTD) (12,52,58,60). Both of these enzymatic activities are implicated in steps in transcription which occur shortly after initiation of the RNA chain. First, a helicase is required for efficient formation of open complexes and for promoter clearance on linear templates in vitro (20). Second, when paused polymerases resume elongation on several Drosophila genes in vivo, the CTD becomes phosphorylated, suggesting a possible role for TFIIH kinase in regulating elongation (50, 70). Furthermore, inhibitors of the TFIIH kinase inhibit elongation under activated (74), but not basal (57), transcription conditions.In vivo, rate-limiting steps after initiation have been well documented for a number of genes. For example, polymerases stall 20 to 40 bases downstream of the start sites in the Drosophila hsp70 and human c-myc genes (38, 51, 64) and terminate...

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“…The general transcription factors IIF and UX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.Recently, it has been shown that eukaryotic RNA polymerase II, like the prokaryotic polymerase, can pause or terminate both in vivo and in vitro transcription within viral (1,3,16,17,19,24,28,29,32,38) and cellular (4,8,9,15,18) genes and thus modulate downstream gene expression. This mechanism is termed attenuation (39).…”

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The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors.

Krauskopf¹,

Bengal²,

Aloni³

1991

Mol. Cell. Biol.

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We have previously reported that both in vivo and in vitro, RNA polymerase H pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCI concentrations (0.2 to 1.5 M) but not at the standard KCI concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCI, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or HeLa cells. The general transcription factors IIF and UX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.Recently, it has been shown that eukaryotic RNA polymerase II, like the prokaryotic polymerase, can pause or terminate both in vivo and in vitro transcription within viral (1,3,16,17,19,24,28,29,32,38) and cellular (4,8,9,15,18) genes and thus modulate downstream gene expression. This mechanism is termed attenuation (39). To date, however, little is known about cis and trans elements involved in the attenuation mechanism in eukaryotes. We have shown that at least in some viral systems, RNA polymerase II can respond to a stem-loop structure in the RNA followed by polyuridines as a signal for the elongation block in vitro (1,3,16,19,28,29,32). In other systems, different sequences constitute the attenuation signal (2,9,31,34,40).One of the model systems in our attenuation studies has been the parvovirus minute virus of mice (MVM). MVM has a genome containing linear single-stranded DNA (5,149 nucleotides [nt]). It is an autonomous virus but is dependent for replication upon cellular functions that are expressed during S phase (10,12,13,36). Transcription initiation on the double-stranded replicative form catalyzed by the host RNA polymerase II has been shown to map to two MVM promoters: P4, located at nt 201 + 5; and P38, located at nt 2005 ± 4 (3, 20, 25).We have previously shown that in vivo and in vitro, RNA polymerase II attenuates transcription at a specific location 142 to 147 nt downstream from the P4 promoter (3, 28). The attenuator RNA was found and mapped in vivo in A9 cells late after infection in both the nuclear and cytoplasmic fractions. Moreover, through the use of in vitro systems, we have shown that the attenuator RNA is a result of pausing or termination and not of processing...

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In vitro formation of short RNA polymerase II transcripts that terminate within the HIV-1 and HIV-2 promoter-proximal downstream regions.

Cited by 122 publications

References 45 publications

Transcriptional arrest of yeast RNA polymerase II by Escherichia coli rho protein in vitro.

Transcriptional arrest of yeast RNA polymerase II by Escherichia coli rho protein in vitro.

Three Functional Classes of Transcriptional Activation Domains

The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors.

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