Stimulation of RNA Polymerase II Elongation by Hepatitis Delta Antigen

Yamaguchi, Yuki; Filipovska, Julija; Yano, Keiichi; Furuya, Akiko; Inukai, Naoto; Narita, Takashi; Sugimoto, Seiji; Konarska, Maria M.; Handa, Hiroshi

doi:10.1126/science.1057925

Cited by 146 publications

(144 citation statements)

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“…Extracts of HeLa cell nuclei were prepared as described (40). Recombinant HDAg (18) and TFIIF (41,42) were prepared as described.…”

Section: Methodsmentioning

confidence: 99%

“…In the viral life cycle, HDAg is thought to stimulate HDV replication and transcription by helping to convert cellular RNAP II from a DNA template-directed RNAP to an RNA template-directed RNAP that can recognize the HDV genome (20 -22). Because HDV replication and transcription are not yet fully recapitulated using in vitro systems (18,23), our laboratory has pursued studies of HDAg stimulation of RNAP II using DNA templates (5,18). In the presence of HDAg, the RNAP II elongation mechanism is significantly altered (5).…”

mentioning

confidence: 99%

“…HDAg participates in HDV replication, in part by stimulating ribozyme activities of HDV RNA (16,17). Recently, however, HDAg was shown to be a potent stimulator of elongation by RNAP II (5,18,19). In the viral life cycle, HDAg is thought to stimulate HDV replication and transcription by helping to convert cellular RNAP II from a DNA template-directed RNAP to an RNA template-directed RNAP that can recognize the HDV genome (20 -22).…”

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confidence: 99%

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Combinatorial Control of Human RNA Polymerase II (RNAP II) Pausing and Transcript Cleavage by Transcription Factor IIF, Hepatitis δ Antigen, and Stimulatory Factor II

Zhang

Yan

Burton

2003

Journal of Biological Chemistry

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When RNA polymerase II (RNAP II) is forced to stall, elongation complexes (ECs) are observed to leave the active pathway and enter a paused state. Initially, ECs equilibrate between active and paused conformations, but with stalls of a long duration, ECs backtrack and become sensitive to transcript cleavage, which is stimulated by the EC rescue factor stimulatory factor II (TFIIS/SII). In this work, the rates for equilibration between the active and pausing pathways were estimated in the absence of an elongation factor, in the presence of hepatitis ␦ antigen (HDAg), and in the presence of transcription factor IIF (TFIIF), with or without addition of SII. Rates of equilibration between the active and paused states are not very different in the presence or absence of elongation factors HDAg and TFIIF. SII facilitates escape from stalled ECs by stimulating RNAP II backtracking and transcript cleavage and by increasing rates into and out of the paused EC. TFIIF and SII cooperate to merge the pausing and active pathways, a combinatorial effect not observed with HDAg and SII. In the presence of HDAg and SII, pausing is observed without stimulation of transcript cleavage, indicating that the EC can pause without backtracking beyond the pretranslocated state.Our laboratory has applied transient state kinetic analysis (1-3) to probe the mechanism and regulation of elongation by human RNA polymerase II (RNAP II) 1 (4, 5). In the current work, we concentrate on control of the branch point between the active synthesis pathway and the pausing pathway. We measure the rate by which RNAP II leaves the active synthesis pathway to enter the pausing pathway after encountering a barrier to elongation caused by withholding the next substrate nucleoside triphosphate.It has been suggested that transcription factor IIF (TFIIF) stimulates RNAP II elongation by suppressing transcriptional pausing (6 -8). TFIIF is a general initiation and elongation factor for RNAP II, composed of RAP74 and RAP30 subunits (RAP for RNA polymerase II-associating protein). In vitro, TFIIF stimulates elongation about 5-10-fold, achieving rates on chromatin-free DNA templates that are similar to rates observed on chromatinized templates in vivo (6 -12). Based on transient state kinetic studies, our laboratory recently proposed a mechanism for RNAP II elongation stimulated by TFIIF and hepatitis ␦ antigen (HDAg) (5). We have also demonstrated the major defects of the RAP74 I176A mutant in general and transient state elongation assays, providing insight into the functions of TFIIF in the RNAP II mechanism (11). We find that TFIIF exerts a global effect on elongation. TFIIF helps commit elongation complexes (ECs) to the forward synthesis pathway. TFIIF stimulates the rate of chemistry and accelerates a slow step after chemistry in the normal processive transition between bonds, a step that includes translocation and pyrophosphate release (5, 11). We conjecture that TFIIF binds to an external surface of RNAP II, tightens the RNAP II clamp, which grasps the RNA-DNA...

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“…Extracts of HeLa cell nuclei were prepared as described (40). Recombinant HDAg (18) and TFIIF (41,42) were prepared as described.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Combinatorial Control of Human RNA Polymerase II (RNAP II) Pausing and Transcript Cleavage by Transcription Factor IIF, Hepatitis δ Antigen, and Stimulatory Factor II

Zhang

Yan

Burton

2003

Journal of Biological Chemistry

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“…The NELF-E subunit has an RNA recognition motif (RRM), which binds RNA, and mutation of the RRM renders NELF unable to repress elongation in vitro (16). Another model, based on the sequence similarity between NELF-A and the hepatitis delta virus antigen posits that the NELF-A subunit associates with the clamp domain of Pol II (20,21). This association could alter the active site of the Pol II in a way that inhibits elongation (1).…”

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confidence: 99%

Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex

Missra

Gilmour

2010

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

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Negative elongation factor (NELF) and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) are involved in pausing RNA Polymerase II (Pol II) in the promoter-proximal region of the hsp70 gene in Drosophila, before heat shock induction. Such blocks in elongation are widespread in the Drosophila genome. However, the mechanism by which DSIF and NELF participate in setting up the paused Pol II remains unclear. We analyzed the interactions among DSIF, NELF, and a reconstituted Drosophila Pol II elongation complex to gain insight into the mechanism of pausing. Our results show that DSIF and NELF require a nascent transcript longer than 18 nt to stably associate with the Pol II elongation complex. Protein-RNA cross-linking reveals that Spt5, the largest subunit of DSIF, contacts the nascent RNA as the RNA emerges from the elongation complex. Taken together, these results provide a possible model by which DSIF binds the elongation complex via association with the nascent transcript and subsequently recruits NELF. Although DSIF and NELF were both required for inhibition of transcription, we did not detect a NELF-RNA contact when the nascent transcript was between 22 and 31 nt long, which encompasses the region where promoter-proximal pausing occurs on many genes in Drosophila. This raises the possibility that RNA binding by NELF is not necessary in promoter-proximal pausing.transcriptional pausing | gene expression | negative elongation factors

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“…Examples include neuronal fate determination during embryonic development (6,44), gene expression of human immunodeficiency virus (5,11,13,19,43), replication and transcription of hepatitis delta virus (38), and transcriptional regulation of heat shock genes (1,10,18). In all these cases, the involvement of three transcription elongation factors, namely, DRB (5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF), NELF (negative elongation factor), and positive transcription elongation factor b (P-TEFb), has been demonstrated or implicated.…”

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confidence: 99%

Human Transcription Elongation Factor NELF: Identification of Novel Subunits and Reconstitution of the Functionally Active Complex

Narita

Yamaguchi

Yano

et al. 2003

Molecular and Cellular Biology

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The elongation step of RNA polymerase II (RNAPII) transcription is emerging as a critical control point for the expression of various genes and for diverse biological processes. Examples include neuronal fate determination during embryonic development (6, 44), gene expression of human immunodeficiency virus (5,11,13,19,43), replication and transcription of hepatitis delta virus (38), and transcriptional regulation of heat shock genes (1,10,18). In all these cases, the involvement of three transcription elongation factors, namely, DRB (5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF), NELF (negative elongation factor), and positive transcription elongation factor b (P-TEFb), has been demonstrated or implicated.Shortly after the initiation of transcription, RNAPII comes under the negative and positive control of DSIF, NELF, and P-TEFb. DSIF and NELF cause transcriptional pausing through physical association with RNAPII. DSIF binds to RNAPII directly and stably (33, 36). However, this appears to have little effect on the catalytic activity of RNAPII (37). A previous study has pointed out that NELF does not bind substantially to DSIF or RNAPII alone but does bind to the complex of DSIF and RNAPII (40). This association is the likely trigger of transcriptional pausing. Conversely, P-TEFb allows RNAPII to enter the productive elongation phase by preventing the action of DSIF and NELF (27, 37). P-TEFb is the protein kinase whose primary target is thought to be the C-terminal domain (CTD) of RNAPII (26). Most, but not all, evidence suggests that P-TEFb-dependent phosphorylation of the CTD facilitates the release of DSIF and NELF from RNA-PII, thereby reversing the inhibition (3, 24, 37). In theory, such regulation at the elongation step allows for rapid change in mRNA levels and for highly sophisticated control over gene expression when combined with regulation at the (pre)initiation step.The structures and functions of DSIF and P-TEFb have been extensively characterized. Human DSIF is a heterodimer composed of p14 (14 kDa) and p160 (160 kDa), whose Saccharomyces cerevisiae counterparts are Spt4 and Spt5 (7,33). In addition to its role in transcriptional pausing, DSIF has a potential to activate RNAPII elongation. The activation mechanism is not well understood: interaction partners of DSIF other than NELF may be involved (13,14,20,23,28). Spt5 has a highly acidic N-terminal region, multiple copies of the KOW motifs, and a repetitive C-terminal region analogous to the RNAPII CTD (9,25,36). RNAPII interacts with Spt5 through a region encompassing the KOW motifs. KOW motifs are also found in the bacterial transcription elongation factor NusG, which binds to prokaryotic RNA polymerase and controls termination and antitermination (15,17,29). In addition, the extreme C terminus of Spt5 is specifically involved in the transcriptional repression pathway (6). Human P-TEFb is a heterodimer composed of Cdk9 (41 kDa) and one of multiple cyclin subunits T1, T2a, T2b, and K (50 to 90 kDa) (26). The k...

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Stimulation of RNA Polymerase II Elongation by Hepatitis Delta Antigen

Cited by 146 publications

References 23 publications

Combinatorial Control of Human RNA Polymerase II (RNAP II) Pausing and Transcript Cleavage by Transcription Factor IIF, Hepatitis δ Antigen, and Stimulatory Factor II

Combinatorial Control of Human RNA Polymerase II (RNAP II) Pausing and Transcript Cleavage by Transcription Factor IIF, Hepatitis δ Antigen, and Stimulatory Factor II

Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex

Human Transcription Elongation Factor NELF: Identification of Novel Subunits and Reconstitution of the Functionally Active Complex

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