[22] RNA polymerase III and class III transcription factors from Saccharomyces cerevisiae

Huet, Janine; Manaud, Nathalie; Dieci, Giorgio; Peyroche, Gérald; Conesa, Christine; Ruet, Anny; Riva, Michel; Sentenac, André

doi:10.1016/s0076-6879(96)73024-0

Cited by 48 publications

(69 citation statements)

References 55 publications

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“…Because the crude Bdp1 fraction (26,27) containing the start site selectivity factor also contains significant levels of DNA-binding proteins (13), we first examined whether a nonspecific DNA-binding protein might impose greater stringency on TFIIIC-dependent placement of TFIIIB. The HMG1-related Nhp6a was chosen to mimic the effect of nonspecific DNA-binding proteins present in crude Bdp1 fractions.…”

Section: Resultsmentioning

confidence: 99%

Nhp6 Is a Transcriptional Initiation Fidelity Factor for RNA Polymerase III Transcription in Vitro and in Vivo

Kassavetis

Steiner

2006

Journal of Biological Chemistry

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The binding of the RNA polymerase III (pol III) transcription factor TFIIIC to the box A intragenic promoter element of tRNA genes specifies the placement of TFIIIB on upstream-lying DNA. In turn, TFIIIB recruits pol III to the promoter and specifies transcription initiating 17-19 base pairs upstream of box A. The resolution of the pol III transcription apparatus into recombinant TFIIIB, highly purified TFIIIC, and pol III is accompanied by a loss of precision in specifying where transcription initiation occurs due to heterogeneous placement of TFIIIB. In this paper we show that Nhp6a, an abundant high mobility group B (HMGB) family, non-sequencespecific DNA-binding protein in Saccharomyces cerevisiae restores transcriptional initiation fidelity to this highly purified in vitro system. Restoration of initiation fidelity requires the presence of Nhp6a prior to TFIIIB-DNA complex formation. Chemical nuclease footprinting of TFIIIC-and TFIIIB-TFIIIC-DNA complexes reveals that Nhp6a markedly alters the TFIIIC footprint over box A and reduces the size of the TFIIIB footprint on upstream DNA sequence. Analyses of unprocessed tRNAs from yeast lacking Nhp6a and its closely related paralogue Nhp6b demonstrate that Nhp6 is required for transcriptional initiation fidelity of some but not all tRNA genes, in vivo.The sites at which RNA polymerase III (pol III) 2 initiates tRNA gene transcription are specified by sequence-specific interactions of its two transcription factors, TFIIIC and TFIIIB, and by pol III itself. TFIIIC is a 520-kDa protein, containing six distinct subunits arranged into two globular subdomains, A (subunits Tfc1, Tfc4, and Tfc7) and B (subunits Tfc3, Tfc6, and Tfc8), which are connected by a flexible linker (Tfc8; see Refs. 1-3 for reviews). TFIIIC binds to two intragenic sequence elements, the start site-proximal box A (binding A) and the distal box B (binding B), which are optimally separated by 30 -60 bp. The B-box B interaction contributes nearly all of the affinity of TFIIIC for tRNA genes (K D Ͻ1 nM) (4). Nevertheless, it is the weak A-box A interaction that primarily specifies initiation 17-19 bp upstream of box A (5, 6). Specification is indirect; the A-box A interaction determines the placement of TFIIIB, which, in turn, recruits pol III. TFIIIB placement is mediated through an interaction between Tfc4 and the Brf1 subunit of TFIIIB. Brf1 forms a stable complex with TBP, and the major DNA contacts during TFIIIC-dependent assembly of TFIIIB upstream of the start site of transcription are mediated by TBP. Consistent with this role of TBP, the DNA segments occupied by TFIIIB (ϳbp Ϫ40 to bp Ϫ10, relative to predicted start sites of transcription) are uniformly AT-rich (Ͼ70% (7-9)).The notion that preferential sequence selection by TBP can occur during TFIIIC-dependent assembly of TFIIIB comes from in vivo and in vitro studies in which a shift in placement of a TATA box or partial TATA box shifts the start site accordingly but retains dependence on TFIIIC for transcription (10, 11). Pol III also cont...

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Section: Resultsmentioning

confidence: 99%

Nhp6 Is a Transcriptional Initiation Fidelity Factor for RNA Polymerase III Transcription in Vitro and in Vivo

Kassavetis

Steiner

2006

Journal of Biological Chemistry

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“…Purification of Pol III and class III transcription factors was described in ref. 44. Pol IIIwt was purified from the yMR4 strain, whereas Pol III⌬ was purified from either C37HA-Ct or yEL7 strain (26).…”

Section: Methodsmentioning

confidence: 99%

Selectivity and proofreading both contribute significantly to the fidelity of RNA polymerase III transcription

Alic

Ayoub²,

Landrieux³

et al. 2007

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

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We examine here the mechanisms ensuring the fidelity of RNA synthesis by RNA polymerase III (Pol III). Misincorporation could only be observed by using variants of Pol III deficient in the intrinsic RNA cleavage activity. Determination of relative rates of the reactions producing correct and erroneous transcripts at a specific position on a tRNA gene, combined with computational methods, demonstrated that Pol III has a highly efficient proofreading activity increasing its transcriptional fidelity by a factor of 10 3 over the error rate determined solely by selectivity (1.8 ؋ 10 ؊4 ). We show that Pol III slows down synthesis past a misincorporation to achieve efficient proofreading. We discuss our findings in the context of transcriptional fidelity studies performed on RNA Pols, proposing that the fidelity of transcription is more crucial for Pol III than Pol II.nucleotide selectivity ͉ transcriptional fidelity

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“…Nuclear extract input, and the column fractions were resolved on 4 -20% SDS-PAGE and analyzed by immunoblotting with antibodies against RNAPII (8WG16) and CDK2. CDK2 and RNAPII coeluted in fractions [15][16][17][18][19]. I, input; rCDK2, recombinant CDK2.…”

Section: Discussionmentioning

confidence: 99%

HIV-1 Tat Interaction with RNA Polymerase II C-terminal Domain (CTD) and a Dynamic Association with CDK2 Induce CTD Phosphorylation and Transcription from HIV-1 Promoter

Deng

Ammosova

Pumfery

et al. 2002

Journal of Biological Chemistry

107

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Human immunodeficiency virus, type 1 (HIV-1), Tat protein activates viral gene expression through promoting transcriptional elongation by RNA polymerase II (RNAPII). In this process Tat enhances phosphorylation of the C-terminal domain (CTD) of RNAPII by activating cell cycle-dependent kinases (CDKs) associated with general transcription factors of the promoter complex, specifically CDK7 and CDK9. We reported a Tat-associated T-cell-derived kinase, which contained CDK2. Here, we provide further evidence that CDK2 is involved in Tat HIV-11 Tat is a viral protein that interacts with transactivation response (TAR) RNA, a hairpin-loop structure at the 5Ј-end of all nascent viral transcripts (reviewed in Refs. 1 and 2). Tat stimulates transcriptional elongation (3), which is regulated by phosphorylation of the largest subunit of RNA polymerase II (RNAPII). The C-terminal domain (CTD) of RNA-PII consists of heptapeptide YSPTSPS repeats, which are phosphorylated on Ser-2 and Ser-5 residues during transcription (reviewed in Ref. 4). CTD is phosphorylated by host cell cycle-dependent protein kinases CDK7 and CDK9 (reviewed in Ref. 5). General transcription factor TFIIH-associated CDK7 phosphorylates Ser-5 during initiation of transcription, whereas positive transcription elongation factor b-associated CDK9 phosphorylates Ser-2 during elongation of transcription (5). Tat associates with the bulge of TAR RNA and also binds to cyclin T1, a cyclin partner of CDK9, which in turn interacts with the loop of TAR RNA (6). This allows CDK9/cyclin T1 to be recruited by Tat to the HIV-1 promoter (7). Tat also modifies the substrate specificity of CDK9 to achieve additional Ser-5 phosphorylation (8).Although the evidence for the role of CDK9/cyclin T1 in Tat-mediated HIV-1 transcription is overwhelming, our recent data suggest that there may be an additional CTD kinase involved in the Tat response. We have purified a Tat-associated T-cell-derived kinase (TTK) that phosphorylates CTD (9 -11). TTK stimulates Tat transactivation in vitro (11) and in vivo (10, 11). TTK containes CDK2, which phosphorylates CDK7 (11).In the work presented here, we analyze the effect of Tat on CTD phosphorylaton by CDK2/cyclin E and the function of CDK2 in transcription assays of HIV-1 promoter in vitro. Our finding demonstrated that interaction of Tat with CTD and a dynamic interaction with CDK2/cyclin E stimulated CTD phosphorylation by CDK2. Also we showed that CDK2 was part of transcription complex and was required for Tat-dependent transcription in vitro.

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[22] RNA polymerase III and class III transcription factors from Saccharomyces cerevisiae

Cited by 48 publications

References 55 publications

Nhp6 Is a Transcriptional Initiation Fidelity Factor for RNA Polymerase III Transcription in Vitro and in Vivo

Nhp6 Is a Transcriptional Initiation Fidelity Factor for RNA Polymerase III Transcription in Vitro and in Vivo

Selectivity and proofreading both contribute significantly to the fidelity of RNA polymerase III transcription

HIV-1 Tat Interaction with RNA Polymerase II C-terminal Domain (CTD) and a Dynamic Association with CDK2 Induce CTD Phosphorylation and Transcription from HIV-1 Promoter

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