Regulation of eukaryotic ribosomal RNA transcription by RNA polymerase modification

Bateman, Erik; Paule, Marvin R.

doi:10.1016/0092-8674(86)90601-x

Cited by 88 publications

(58 citation statements)

References 20 publications

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“…It is not caused by incubation of the reaction per se, because an additional preincubation of 1 supplemented with factor C, the initiation-competent form of RNA polymerase I (lane 19). To further delineate the activating component, factor C was dissociated into pol (an elongation-competent but specific initiation-deficient RNA polymerase I) and factor C* (which provides the specific initiation capacity to pol but lacks polymerase activity itself [see below]).…”

Section: Resultsmentioning

confidence: 99%

“…Finally, in apparent analogy to the loss of factor C activity in growth-arrested mammalian cells, encysting cells of the soil amoeba Acanthamoeba castellandi also demonstrate downregulation of rRNA gene transcription as a result of loss of specific initiation competence by the RNA polymerase I (1,48). No specific initiation factor has been separated from the A. castellandi RNA polymerase I catalytic activity, but the A. castellanii RNA polymerase I (which has twice as many copurifying polypeptides as does the mammalian enzyme) could bind its C* equivalent more tightly.…”

Section: -Nt)mentioning

confidence: 98%

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Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process.

Brun¹,

Ryan²,

Sollner‐Webb³

1994

Mol. Cell. Biol.

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Factor C* is the component of the RNA polymerase I holoenzyme (factor C) that allows specific transcriptional initiation on a factor D (SL1)- and UBF-activated rRNA gene promoter. The in vitro transcriptional capacity of a preincubated rDNA promoter complex becomes exhausted very rapidly upon initiation of transcription. This is due to the rapid depletion of C* activity. In contrast, C* activity is not unstable in the absence of transcription, even in the presence of nucleoside triphosphates (NTPs). By using 3'dNTPs to specifically halt elongation, C* is seen to remain active through transcription complex assembly, initiation, and the first approximately 37 nucleotides of elongation, but it is inactivated before synthesis proceeds beyond approximately 40 nucleotides. When elongation is halted before this critical distance, the C* remains active and on that template complex, greatly extending the kinetics of transcription and generating manyfold more transcripts than would have been synthesized if elongation had proceeded past the critical distance where C* is inactivated. In complementary in vivo analysis under conditions where C* activity is not replenished, C* activity becomes depleted from cells, but this also occurs only when there is ongoing rDNA transcription. Thus, both in vitro and in vivo, the specific initiation-conferring component of the RNA polymerase I holoenzyme is used stoichiometrically in the transcription process.

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Section: Resultsmentioning

confidence: 99%

Section: -Nt)mentioning

confidence: 98%

Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process.

Brun¹,

Ryan²,

Sollner‐Webb³

1994

Mol. Cell. Biol.

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“…In previous reports, various aspects of the initiation complex were described (1,2,14,15,16). In the work described in this report, we combined several approaches to study more (pH 5.2), and tRNA to final concentrations of 0.1%, 0.3 M, and 100 ,ug/ml, respectively.…”

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confidence: 99%

“…Nonetheless DNA or protein conformational changes accompany elongation by RNA polymerase I. RNA polymerase I footprints following transcription elongation were obtained by adding either ATP, GTP, and 3'-O-methyl CTP or ATP, GTP, CTP, and UTP to preformed TIF-polymerase DNA complexes and then digesting with MPE-Fe(II) in the usual way. Addition of ATP, GTP, and 3'-O-methyl CTP allows elongation to nucleotide +8, where further elongation is blocked (2). Addition of all four nucleotides allows unimpeded elongation and, presumably, reinitiation.…”

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confidence: 99%

Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates.

Bateman

Paule

1988

Mol. Cell. Biol.

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Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.Transcription of the Acanthamoeba castellanii rRNA gene in vitro requires a single transcription initiation factor (TIF) and RNA polymerase I (1,14,17). The properties of this system broadly resemble those of other eucaryotic genes (3,8,24) in that TIF functions by forming a stable complex with the promoter and directs RNA polymerase I to accurately initiate transcription (1,14). The promoter of the Acanthamoeba rRNA gene is relatively simple, consisting of an A+T-rich sequence around the transcription start site, which is required for polymerase function, and a second region extending from -20 to -47 (14, 17). This second region is required for TIF binding and can be divided into two portions. One, roughly between -32 and -47, affects the stability of TIF-DNA interactions, and the other, between -31 and -20, is absolutely required for transcription (14,17 (20,23).In previous reports, various aspects of the initiation complex were described (1, 2, 14, 15, 16). In the work described in this report, we combined several approaches to study more dynamic aspects of preinitiation, initiation, and elongating complexes. The results indicate that transcription initiation is accompanied by conformational changes in the polymerase-DNA complex as the enzyme translocates.These changes lead to the conversion of an apparently closed complex to an open complex. Polymerase becomes spatially separated from promoter-bound TIF and covers an unwound DNA region of at least 10 base pairs. MATERIALS AND METHODSProteins and templates. TIF and RNA polymerase I were prepared from A. castellanii as described previously (1). For some experiments, both TIF and RNA polymerase I were further purified on 10 to 30% sucrose gradients to remove contaminating topoisomerase or catenase activities. Following sedimentation, RNA polymerase I was essentially homogeneous, but TIF still contained several bands on sodium dodecyl sulfate gels. Escherichia coli RNA polymerase was the kind gift of A. Y. Woody of our department. The enzyme was more than 95% pure and contained 90% hol...

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“…Remarkably, both Ϫ12 and Ϫ6 are outside the region footprinted by the fundamental transcription initiation factor, TIF-IB, from A. castellanii (21,22). However, this region does exhibit enhanced bands in some footprinting experiments, and point mutations in this region affect transcription efficiency (11).…”

Section: Resultsmentioning

confidence: 99%

Identification of Previously Unrecognized Common Elements in Eukaryotic Promoters

Radebaugh

Gong

Bartholomew

et al. 1997

Journal of Biological Chemistry

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A new ribosomal RNA promoter element with a functional role similar to the RNA polymerase II initiator (Inr) was identified. This sequence, which we dub the ribosomal Inr (rInr) is unusually conserved, even in normally divergent RNA polymerase I promoters. It functions in the recruitment of the fundamental, TATA-binding protein (TBP)-containing transcription factor, TIF-IB. All upstream elements of the exceptionally strong Acanthamoeba castellanii ribosomal RNA core promoter, to within 6 base pairs of the transcription initiation site (tis), can be deleted without loss of specific transcription initiation. Thus, the A. castellanii promoter can function in a manner similar to RNA polymerase II TATA-less promoters. Sequence-specific photocross-linking localizes a 96-kDa subunit of TIF-IB and the second largest RNA polymerase I subunit (A 133 ) to the rInr sequence. A 185 also photo-cross-links when polymerase is stalled at ؉7.Because promoters for some small nuclear RNA genes switch between polymerase II and III as a result of simple sequence/ spacing alterations, promoters for eukaryotic RNA polymerases II and III are considered more similar to each other than to the RNA polymerase I promoter (reviewed in Ref. 1). Most polymerase II promoters contain a TATA box, or an initiator element (Inr), 1 or both (2). The Inr surrounds the transcription initiation site (tis) and the TATA box is upstream about 30 base pairs. The TATA box is the specific binding site for TATA-binding protein (TBP), the subunit common to the fundamental transcription factors (3) for all polymerases (4). On polymerase II genes with TATA boxes, TBP alone can nucleate the assembly of an initiation complex. In contrast, TATA-less promoters require additional factors or TFIID subunits, called TBP-associated factors (TAFs) (5). Drosophila TAF II 150 (dTAF II 150) specifically interacts with sequences including the Inr, tethering TBP to the promoter (6), and nucleating the assembly of the initiation complex. In humans, the functional dTAF II 150 homolog is CIF and is not tightly associated with TFIID (7). Promoters with both sequence elements are unusually strong, because they bind TFIID very efficiently.The Acanthamoeba castellanii rRNA core promoter is also unusually strong, binding TIF-IB with a dissociation constant of approximately 30 pM. 2 We noted that this promoter, along with the promoters for other rRNA genes, contains a conserved sequence element which surrounds the tis (8). This is the only well-conserved sequence in eukaryotic rRNA promoters. In addition, Windle and Sollner-Webb (9) observed that in Xenopus laevis oocytes injected with huge quantities (approximately 1 ϫ 10 7 copies) of plasmid ribosomal DNA, the minimal rRNA promoter only encompassed this conserved sequence, although this seemed to be unique to this system and unusual assay condition. In Arabidopsis thaliana, mutations in the conserved sequence in the context of the full-length rRNA promoter decrease transcription in transient expression assays (10). However, the conserv...

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Regulation of eukaryotic ribosomal RNA transcription by RNA polymerase modification

Cited by 88 publications

References 20 publications

Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process.

Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process.

Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates.

Identification of Previously Unrecognized Common Elements in Eukaryotic Promoters

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