Purification of a factor from Ehrlich ascites tumor cells specifically stimulating RNA polymerase II

Sekimizu, Kazuhisa; Kobayashi, Nobuyuki; Mizuno, Den′ichi; Natori, Shunji

doi:10.1021/bi00668a018

Cited by 98 publications

(60 citation statements)

References 19 publications

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“…Antitermination by the N protein involves the host proteins, NusA, NusB, NusG, ribosomal protein S10 and RNA elements at the 5Ј end of nascent viral transcripts. Whereas processive mechanisms found in phage have been extensively characterized and references therein), the mechanisms in eukaryotes are much less understood, except for the Drosophila hsp70 gene (Rougvie and Lis 1988;O'Brien and Lis 1991), the c-myc gene (Krumm et al 1992;Strobl and Eick 1992), and factor TFIIF (Price et al 1989;Bengal et al 1991), TFIIS (Sekimizu et al 1976), SIII (Elongin) (Bradsher et al 1993a,b;Aso et al 1995), P-TEFb (Marshall andPrice 1995;Marshall et al 1996), and HIV-1 Tat (Marciniak and Sharp 1991;Kato et al 1992;Zhou and Sharp 1995;Mancebo et al 1997;Zhu et al 1997). Tat activation of HIV-1 transcription is interesting because Tat enhances the processivity of transcription complexes in a manner reminiscent of N , and Tat activation is sensitive to DRB (Marciniak and Sharp 1991;Zhou and Sharp 1995;Mancebo et al 1997;Zhu et al 1997).…”

Section: Antitermination Mechanisms In Eukaryotesmentioning

confidence: 99%

DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs

Wada

Takagi

Yamaguchi

et al. 1998

Genes & Development

657

707

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We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB). This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits. Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5. Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates. A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.

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Section: Antitermination Mechanisms In Eukaryotesmentioning

confidence: 99%

DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs

Wada

Takagi

Yamaguchi

et al. 1998

Genes & Development

657

707

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“…TFIIS, the first eukaryotic elongation factor identified, was isolated nearly three decades ago based on its ability to induce long transcripts in an in vitro system (62). Further analysis of TFIIS revealed that it helps elongation in vitro by stimulating an RNA transcript cleavage activity of Pol II (13,23).…”

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confidence: 99%

Identification and Characterization of Elf1, a Conserved Transcription Elongation Factor in Saccharomyces cerevisiae

Prather

Krogan

Emili

et al. 2005

Molecular and Cellular Biology

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In order to identify previously unknown transcription elongation factors, a genetic screen was carried out to identify mutations that cause lethality when combined with mutations in the genes encoding the elongation factors TFIIS and Spt6. This screen identified a mutation in YKL160W, hereafter named ELF1 (elongation factor 1). Further analysis identified synthetic lethality between an elf1⌬ mutation and mutations in genes encoding several known elongation factors, including Spt4, Spt5, Spt6, and members of the Paf1 complex. Genome-wide synthetic lethality studies confirmed that elf1⌬ specifically interacts with mutations in genes affecting transcription elongation. Chromatin immunoprecipitation experiments show that Elf1 is cotranscriptionally recruited over actively transcribed regions and that this association is partially dependent on Spt4 and Spt6. Analysis of elf1⌬ mutants suggests a role for this factor in maintaining proper chromatin structure in regions of active transcription. Finally, purification of Elf1 suggests an association with casein kinase II, previously implicated in roles in transcription. Together, these results suggest an important role for Elf1 in the regulation of transcription elongation.Eukaryotic transcription is a complex process consisting of a series of steps involving initiation, elongation, and termination. Many recent studies have revealed the extent to which these steps are linked to each other, both functionally and physically (54, 71). While transcription initiation has been well studied and many of the fundamental mechanisms have been identified, an understanding of the control of transcription elongation in vivo is less clear. Some understanding of elongation has come from in vitro experiments examining the effects of various factors in modulating the elongation properties of RNA polymerase II (Pol II) (66). In the past few years many studies have also started to address the control of elongation in vivo and have identified many additional factors believed to play a role in this process (3,69). In this paper, we describe a previously unstudied factor, Elf1 of Saccharomyces cerevisiae, that is functionally related to several other elongation factors and complexes, including TFIIS, Spt6, and the Paf1 complex.TFIIS, the first eukaryotic elongation factor identified, was isolated nearly three decades ago based on its ability to induce long transcripts in an in vitro system (62). Further analysis of TFIIS revealed that it helps elongation in vitro by stimulating an RNA transcript cleavage activity of Pol II (13, 23). This activity allows backtracked (arrested) Pol II to cleave the 3Ј end of the nascent transcript, thus realigning the 3Ј end of the transcript with the Pol II catalytic site. Additional evidence suggests that TFIIS can help Pol II overcome a promoterproximal pause (1, 50). Finally, recent evidence has shown that TFIIS is recruited along transcribed open reading frames, further suggesting a general role in elongation (51, 53).Spt6 is one member of a class of elongat...

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“…Two cellular factors, TFIIF (also referred as RAP30/74, P7, and FC) (Sopta et al 1985;Conaway and Conaway 1989;Flores et al 1990;Kitajima et al 1990) and TFIIS (Sekimizu et al 1976), have been reported to enhance transcriptional elongation in vitro (Reinberg and Roeder 1987;Flores et al 1989). It was reported that TFIIS suppresses pausing and also releases the paused complex, whereas TFIIF suppresses only pausing with an increased rate of elongation (Bengal et al 1991).…”

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confidence: 99%

HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors.

Sumimoto²,

et al. 1992

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The HIV-1 trans-activator Tat increases the rate of transcription from the HIV-1 LTR promoter through the stem-loop-containing TAR RNA. To analyze the mechanisms of Tat action, a cell-free trans-activation system with no preincubation has been developed. Recombinant Tat specifically increased the level of a long runoff transcript but not a promoter-proximal transcript in a TAR-dependent fashion. These observations and the result of pulse-chase experiments support strongly the hypothesis that Tat enhances the ability of RNA polymerase to elongate over longer distances. Increased levels of the purified cellular factor TFIIF, essential for initiation and also implicated in elongation of transcription, obviated tr^ins-activation by Tat by increasing the basal (Tat-independent) activity. However, another elongation factor, ATN/TFIIS, showed synergistic activation with Tat. An antiserum against a recombinant form of the large subunit of TFIIF (RAP 74) preferentially suppressed the activated level of transcription exerted by Tat. We propose the hypothesis that Tat acts as a processivity factor on RNA polymerase II in an analogous manner to TFIIF.

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Purification of a factor from Ehrlich ascites tumor cells specifically stimulating RNA polymerase II

Cited by 98 publications

References 19 publications

DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs

DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs

Identification and Characterization of Elf1, a Conserved Transcription Elongation Factor in Saccharomyces cerevisiae

HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors.

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