Transcription Elongation through DNA Arrest Sites

Awrey, Donald E.; Weilbaecher, Rodney G.; Hemming, Sally A.; Orlicky, Stephen; Kane, Caroline M.; Edwards, A.M.

doi:10.1074/jbc.272.23.14747

Cited by 105 publications

(78 citation statements)

References 53 publications

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“…elongation at the level of the cleaving RNase activity (9). This elongation defect is consistent with the strong sensitivity of rpb9 null mutants to nucleotide-depleting drugs (15).…”

Section: Rpb9 Contribution To Tfiie Recruitment On Rna Polymerase IIsupporting

confidence: 60%

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The Rpb9 Subunit of RNA Polymerase II Binds Transcription Factor TFIIE and Interferes with the SAGA and Elongator Histone Acetyltransferases

Mullem

Wéry

Werner³

et al. 2002

Journal of Biological Chemistry

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Rpb9 is a small subunit of yeast RNA polymerase II participating in elongation and formed of two conserved zinc domains. rpb9 mutants are viable, with a strong sensitivity to nucleotide-depleting drugs. Deleting the C-terminal domain down to the first 57 amino acids has no detectable growth defect. Thus, the critical part of Rpb9 is limited to a N-terminal half that contacts the lobe of the second largest subunit (Rpb2) and forms a ␤-addition motif with the "jaw" of the largest subunit (Rpb1). Rpb9 has homology to the TFIIS elongation factor, but mutants inactivated for both proteins are indistinguishable from rpb9 single mutants. In contrast, rpb9 mutants are lethal in cells lacking the histone acetyltransferase activity of the RNA polymerase II Elongator and SAGA factors. In a two-hybrid test, Rpb9 physically interacts with Tfa1, the largest subunit of TFIIE. The interacting fragment, comprising amino acids 62-164 of Tfa1, belongs to a conserved zinc motif. Tfa1 is immunoprecipitated by RNA polymerase II. This co-purification is strongly reduced in rpb9-⌬, suggesting that Rpb9 contributes to the recruitment of TFIIE on RNA polymerase II.The recent determination of the bacterial RNA polymerase (1) and yeast RNA polymerase II (Pol II) 1 (2) core structures has opened a new chapter in transcription studies. The remarkably similar organization of these two enzymes leaves no doubt as to the strong mechanistic conservation of the transcription process. This similarity was anticipated from the sequence homology existing between the ␤Ј␤␣ 2 bacterial core enzyme and 5 of the 10 core subunits of Pol II. Rpb1 and Rpb2 are homologues of ␤Ј and ␤, the Rpb3/Rpb11 heterodimer corresponding to the bacterial ␣2 dimer, and is distantly related to Rpb6 (3). On the other hand, Pol II contains five small subunits (Rpb5, Rpb8, Rpb9, Rpb10, Rpb12) not found in the bacterial enzyme. These subunits also belong to the core structure of Pol I and Pol III or are related to it, in the case of Rpb9. Except for Rpb8, they are also akin to archaeal polypeptides (4, 5).The present study deals with the Rpb9 subunit, belonging to a conserved family of eukaryotic and archeal zinc-binding polypeptides that also includes the yeast Pol I (Rpa12 (6)) and Pol III (Rpc11 (7)) subunits and the TFIIS elongation factor (8 -10) encoded by PPR2 in Saccharomyces cerevisiae (11,12). There is ample evidence that Rpb9 (9), Rpc11 (7), and TFIIS (9, 13) control transcription elongation by activating the RNA cleavage activity inherent to all RNA polymerases. The fact that yeast ppr2 (14), rpb9 (15), and rpa12 (16) mutants are strongly sensitive to nucleotide-depleting drugs is also consistent with a major elongation defect. Rpb9 is highly conserved in evolution, and the yeast subunit can be replaced in vivo by its human counterpart (17). However, animal mutants (Drosophila melanogaster) are lethal (18), whereas yeast null mutants have only a limited growth defect (19).Rpa12, Rpb9, and Rpc11 have a related organization in Pol I, Pol II, and Pol III, respectively. ...

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“…elongation at the level of the cleaving RNase activity (9). This elongation defect is consistent with the strong sensitivity of rpb9 null mutants to nucleotide-depleting drugs (15).…”

Section: Rpb9 Contribution To Tfiie Recruitment On Rna Polymerase IIsupporting

confidence: 60%

“…There is ample evidence that Rpb9 (9), Rpc11 (7), and TFIIS (9,13) control transcription elongation by activating the RNA cleavage activity inherent to all RNA polymerases. The fact that yeast ppr2 (14), rpb9 (15), and rpa12 (16) mutants are strongly sensitive to nucleotide-depleting drugs is also consistent with a major elongation defect.…”

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confidence: 99%

The Rpb9 Subunit of RNA Polymerase II Binds Transcription Factor TFIIE and Interferes with the SAGA and Elongator Histone Acetyltransferases

Mullem

Wéry

Werner³

et al. 2002

Journal of Biological Chemistry

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“…The mutant polymerase, RPII⌬9, possesses "wild type" intrinsic cleavage activity (this work) and binds TFIIS efficiently, yet RPII⌬9 responds poorly to TFIIS as assayed by either cleavage or readthrough (22). These results suggested that Rpb9p transmits the signal from TFIIS to the active site of the polymerase rather than impacting the intrinsic ability of the polymerase to cleave the nascent transcript.…”

Section: Discussionmentioning

confidence: 64%

“…For yeast RNA polymerase II (RPII), the Rpb9p subunit has been shown to mediate the signal between TFIIS and the polymerase catalytic center for stimulating transcript cleavage (22). Arrested ternary complexes formed with yeast RNA polymerase II lacking Rpb9 (RPII⌬9) are much less responsive to TFIIS, and yet they can carry out the intrinsic cleavage reaction.…”

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confidence: 99%

Intrinsic Transcript Cleavage in Yeast RNA Polymerase II Elongation Complexes

Weilbaecher

Awrey

Edwards

et al. 2003

Journal of Biological Chemistry

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Transcript elongation can be interrupted by a variety of obstacles, including certain DNA sequences, DNAbinding proteins, chromatin, and DNA lesions. Bypass of many of these impediments is facilitated by elongation factor TFIIS through a mechanism that involves cleavage of the nascent transcript by the RNA polymerase II/TFIIS elongation complex. Highly purified yeast RNA polymerase II is able to perform transcript hydrolysis in the absence of TFIIS. The "intrinsic" cleavage activity is greatly stimulated at mildly basic pH and requires divalent cations. Both arrested and stalled complexes can carry out the intrinsic cleavage reaction, although not all stalled complexes are equally efficient at this reaction. Arrested complexes in which the nascent transcript was cleaved in the absence of TFIIS were reactivated to readthrough blocks to elongation. Thus, cleavage of the nascent transcript is sufficient for reactivating some arrested complexes. Small RNA products released following transcript cleavage in stalled ternary complexes differ depending upon whether the cleavage has been induced by TFIIS or has occurred in mildly alkaline conditions. In contrast, both intrinsic and TFIIS-induced small RNA cleavage products are very similar when produced from an arrested ternary complex. Although ␣-amanitin interferes with the transcript cleavage stimulated by TFIIS, it has little effect on the intrinsic cleavage reaction. A mutant RNA polymerase previously shown to be refractory to TFIIS-induced transcript cleavage is essentially identical to the wild type polymerase in all tested aspects of intrinsic cleavage.Gene expression can be controlled by regulating any step of the transcription cycle: promoter binding, initiation, promoter escape, elongation, or termination. After transcript synthesis begins, the ability to suppress or complete the synthesis of an RNA transcript is vital to the cell, and a substantial and growing number of genes have been reported to be regulated during transcript elongation (1).The ternary elongation complex consists of the RNA polymerase, the template DNA, and the nascent RNA transcript. Surratt et al. (2) discovered that bacterial RNA polymerase ternary complexes have a mechanism for transcript shortening, endonucleolytic cleavage near the 3Ј-end of the transcript. After transcript cleavage, the 3Ј-fragment is released while the 5Ј-fragment is retained in an active ternary complex. A single cleavage event can liberate from 1 to 17 nt 1 of RNA bearing a 5Ј-monophosphate (3-6). Remarkably, transcription accurately resumes from the site of transcript cleavage to re-synthesize the excised RNA.Transcript cleavage activity is conserved in many DNA-dependent RNA polymerases, including bacterial RNA polymerases, vaccinia virus RNA polymerase (7), RNA polymerase I (8, 9), RNA polymerase II (10 -14), and RNA polymerase III (15). Accessory factors that stimulate transcript cleavage in ternary complexes have been identified in both prokaryotes (GreA and GreB) and eukaryotes (reviewed in Refs. 3,9,[16][17]...

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“…Furthermore, all three subunits are homologous to TFIIS, a well characterized Pol II elongation factor, further pointing to a common postelongation function (23,26). Because Rpc11p has been directly implicated in Pol III termination (10) and Rpb9p is required for Pol II to respond to pause and arrest sites during elongation (27), we wished to determine whether Rpa12p plays a similar role for Pol I. TRO analysis of the ⌬rpa12 deletion strain shows a dramatic loss of Pol I termination, resulting in high levels of Pol I escaping the Reb1p terminator and reading into the spacer sequence. Electron microscopy (EM) visualization of these spacer Pol I complexes (28) seen with the ⌬rpa12 deletion strain surprisingly reveals they are for the most part devoid of attached nascent RNA.…”

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confidence: 99%

Transcriptional termination by RNA polymerase I requires the small subunit Rpa12p

Prescott

Osheim

Jones

et al. 2004

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

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We identify Rpa12p of RNA polymerase I (Pol I) as a termination factor. Combined analyses using transcription run-on, electron microscopy-visualized chromatin spreading and RT-PCR have been applied to the rRNA-encoding genes of Saccharomyces cerevisiae. These confirm that Pol I termination occurs close to the Reb1p-dependent terminator in wild-type strains. However, deletion mutants for the 3 end-processing enzyme Rnt1p or the Rpa12p subunit of Pol I both show Pol I transcription in the spacer. For ⌬rpa12, these spacer polymerases are devoid of nascent transcripts, suggesting they are immediately degraded. The homology of Rpa12p to the small subunit Rpb9p of Pol II and Rpc11p of Pol III, both implicated in transcriptional termination, points to a common termination mechanism for all three classes of RNA polymerase.

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Transcription Elongation through DNA Arrest Sites

Cited by 105 publications

References 53 publications

The Rpb9 Subunit of RNA Polymerase II Binds Transcription Factor TFIIE and Interferes with the SAGA and Elongator Histone Acetyltransferases

The Rpb9 Subunit of RNA Polymerase II Binds Transcription Factor TFIIE and Interferes with the SAGA and Elongator Histone Acetyltransferases

Intrinsic Transcript Cleavage in Yeast RNA Polymerase II Elongation Complexes

Transcriptional termination by RNA polymerase I requires the small subunit Rpa12p

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