scholarly journals A Step Subsequent to Preinitiation Complex Assembly at the Ribosomal RNA Gene Promoter Is Rate Limiting for Human RNA Polymerase I-Dependent Transcription

2001 ◽  
Vol 21 (8) ◽  
pp. 2641-2649 ◽  
Author(s):  
Kostya I. Panov ◽  
J. Karsten Friedrich ◽  
Joost C. B. M. Zomerdijk
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Pol I ◽  
Initiation Of Transcription ◽  
Polymerase I ◽  
Rate Limiting

ABSTRACT The assembly, disassembly, and functional properties of transcription preinitiation complexes (PICs) of human RNA polymerase I (Pol I) play a crucial role in the regulation of rRNA gene expression. To study the factors and processes involved, an immobilized-promoter template assay has been developed that allows the isolation from nuclear extracts of functional PICs, which support accurate initiation of transcription. Immunoblotting of template-bound factors showed that these complexes contained the factors required to support initiation of transcription, SL1, upstream binding factor (UBF), and Pol I. We have demonstrated that, throughout a single round of transcription, SL1 and UBF remain promoter bound. Moreover, the promoter-bound SL1 and UBF retain the ability to function in transcription initiation. SL1 has a central role in the stable association of the PIC with the promoter DNA. The polymerase component of the PIC is released from the promoter during transcription yet is efficiently recycled and able to reinitiate from “poised” promoters carrying SL1 and UBF, since the PICs captured on the immobilized templates sustained multiple rounds of transcription. Kinetic analyses of initiation of transcription by Pol I revealed that Pol I-dependent transcription is rate limited in a step subsequent to recruitment and assembly of Pol I PICs. The rate of RNA synthesis is primarily determined by the rates at which the polymerase initiates transcription and escapes the promoter, referred to as promoter clearance. This rate-limiting step in Pol I transcription is likely to be a major target in the regulation of rRNA gene expression.

scholarly journals Sequestration analysis for RNA polymerase I transcription factors with various deletion and point mutations reveals different functional regions of the mouse rRNA gene promoter.

1987 ◽  
Vol 7 (4) ◽  
pp. 1486-1495 ◽  
Author(s):  
M Nagamine ◽  
T Kishimoto ◽  
J Aono ◽  
H Kato ◽  
R Kominami ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Cell Extract ◽  
Rna Polymerase I ◽  
Base Substitution ◽  
Rrna Gene ◽  
Null Mutant ◽  
Initiation Reaction ◽  
Polymerase I

We compared the ability of various deletion and substitution mutants of the mouse rRNA gene promoter to bind essential factors required for accurate transcription initiation by RNA polymerase I. Different amounts of a competitor template were first incubated with a mouse cell extract containing the whole complement of factors and RNA polymerase I, and then a tester template was added for the second incubation. Transcription was started by adding nucleoside triphosphates (one labeled), and the accurate transcripts were determined on a gel. The results indicated that the ability of 5' deletion mutants to sequester essential factors decreased almost concurrently with the impairment of in vitro transcription activity, whereas when the promoter sequence was removed from the 3' side, the transcription activity decreased earlier and more drastically than the sequestration ability. Similar, though not identical, results were obtained by preincubation with fraction D separated on a phosphocellulose column, indicating that the major factor which was sequestered was TFID, the species-dependent transcription initiation factor that binds first to the promoter in the initiation reaction (H. Kato, M. Nagamine, R. Kominami, and M. Muramatsu, Mol. Cell. Biol. 6:3418-3427, 1986). Compilation of the data suggests that a region inside the 5' half of the core promoter (-40 to -1) is essential for the binding of TFID. The 3' half of the promoter (-1 to downstream) is not essential for the binding of TFID but is highly important for an efficient transcription initiation. A strong down-mutant with a one-base substitution at -16 (G to A) had a reduced ability to bind to TFID, whereas a null mutant with a single base substitution at -7 (G to A) showed a binding ability similar to that of the wild-type promoter when tested with whole-cell extract. This null mutant, however, could not sequester the TFID well when incubated with fraction D alone, suggesting that the binding of TFID with this mutant is unstable in the absence of another factor(s) present in cell extract. The factor is not TFIA, which binds after TFID, because the addition of fraction A containing TFIA did not cause TFID to bind to the mutant. The availability of different mutants having lesions at different steps of transcription initiation will provide a powerful tool for the dissection of the initiation reaction of the RNA gene.

scholarly journals Phosphorylation by Casein Kinase 2 Facilitates rRNA Gene Transcription by Promoting Dissociation of TIF-IA from Elongating RNA Polymerase I

2008 ◽  
Vol 28 (16) ◽  
pp. 4988-4998 ◽  
Author(s):  
Holger Bierhoff ◽  
Miroslav Dundr ◽  
Annemieke A. Michels ◽  
Ingrid Grummt
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Rna Polymerase I ◽  
Initiation Factor ◽  
Rrna Gene ◽  
Rdna Transcription ◽  
Pol I ◽  
Polymerase I

ABSTRACT The protein kinase casein kinase 2 (CK2) phosphorylates different components of the RNA polymerase I (Pol I) transcription machinery and exerts a positive effect on rRNA gene (rDNA) transcription. Here we show that CK2 phosphorylates the transcription initiation factor TIF-IA at serines 170 and 172 (Ser170/172), and this phosphorylation triggers the release of TIF-IA from Pol I after transcription initiation. Inhibition of Ser170/172 phosphorylation or covalent tethering of TIF-IA to the RPA43 subunit of Pol I inhibits rDNA transcription, leading to perturbation of nucleolar structure and cell cycle arrest. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrate that dissociation of TIF-IA from Pol I is a prerequisite for proper transcription elongation. In support of phosphorylation of TIF-IA switching from the initiation into the elongation phase, dephosphorylation of Ser170/172 by FCP1 facilitates the reassociation of TIF-IA with Pol I, allowing a new round of rDNA transcription. The results reveal a mechanism by which the functional interplay between CK2 and FCP1 sustains multiple rounds of Pol I transcription.

Sequestration analysis for RNA polymerase I transcription factors with various deletion and point mutations reveals different functional regions of the mouse rRNA gene promoter

1987 ◽  
Vol 7 (4) ◽  
pp. 1486-1495
Author(s):  
M Nagamine ◽  
T Kishimoto ◽  
J Aono ◽  
H Kato ◽  
R Kominami ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Cell Extract ◽  
Rna Polymerase I ◽  
Base Substitution ◽  
Rrna Gene ◽  
Null Mutant ◽  
Initiation Reaction ◽  
Polymerase I

We compared the ability of various deletion and substitution mutants of the mouse rRNA gene promoter to bind essential factors required for accurate transcription initiation by RNA polymerase I. Different amounts of a competitor template were first incubated with a mouse cell extract containing the whole complement of factors and RNA polymerase I, and then a tester template was added for the second incubation. Transcription was started by adding nucleoside triphosphates (one labeled), and the accurate transcripts were determined on a gel. The results indicated that the ability of 5' deletion mutants to sequester essential factors decreased almost concurrently with the impairment of in vitro transcription activity, whereas when the promoter sequence was removed from the 3' side, the transcription activity decreased earlier and more drastically than the sequestration ability. Similar, though not identical, results were obtained by preincubation with fraction D separated on a phosphocellulose column, indicating that the major factor which was sequestered was TFID, the species-dependent transcription initiation factor that binds first to the promoter in the initiation reaction (H. Kato, M. Nagamine, R. Kominami, and M. Muramatsu, Mol. Cell. Biol. 6:3418-3427, 1986). Compilation of the data suggests that a region inside the 5' half of the core promoter (-40 to -1) is essential for the binding of TFID. The 3' half of the promoter (-1 to downstream) is not essential for the binding of TFID but is highly important for an efficient transcription initiation. A strong down-mutant with a one-base substitution at -16 (G to A) had a reduced ability to bind to TFID, whereas a null mutant with a single base substitution at -7 (G to A) showed a binding ability similar to that of the wild-type promoter when tested with whole-cell extract. This null mutant, however, could not sequester the TFID well when incubated with fraction D alone, suggesting that the binding of TFID with this mutant is unstable in the absence of another factor(s) present in cell extract. The factor is not TFIA, which binds after TFID, because the addition of fraction A containing TFIA did not cause TFID to bind to the mutant. The availability of different mutants having lesions at different steps of transcription initiation will provide a powerful tool for the dissection of the initiation reaction of the RNA gene.

scholarly journals Stimulation of the mouse rRNA gene promoter by a distal spacer promoter.

1995 ◽  
Vol 15 (8) ◽  
pp. 4648-4656 ◽  
Author(s):  
M H Paalman ◽  
S L Henderson ◽  
B Sollner-Webb
Keyword(s):  
Rna Polymerase ◽  
Gene Promoter ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Transcriptional Terminator ◽  
Wide Range ◽  
Polymerase I ◽  
Enhancer Sequences ◽  
Stimulation Of

We show that the mouse ribosomal DNA (rDNA) spacer promoter acts in vivo to stimulate transcription from a downstream rRNA gene promoter. This augmentation of mammalian RNA polymerase I transcription is observed in transient-transfection experiments with three different rodent cell lines, under noncompetitive as well as competitive transcription conditions, over a wide range of template concentrations, whether or not the enhancer repeats alone stimulate or repress expression from the downstream gene promoter. Stimulation of gene promoter transcription by the spacer promoter requires the rDNA enhancer sequences to be present between the spacer promoter and gene promoter and to be oriented as in native rDNA. Stimulation also requires that the spacer promoter be oriented toward the enhancer and gene promoter. However, stimulation does not correlate with transcription from the spacer promoter because the level of stimulation is not altered by either insertion of a functional mouse RNA polymerase I transcriptional terminator between the spacer promoter and enhancer or replacement with a much more active heterologous polymerase I promoter. Further analysis with a series of mutated spacer promoters indicates that the stimulatory activity does not reside in the major promoter domains but requires the central region of the promoter that has been correlated with enhancer responsiveness in vivo.

Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis

1986 ◽  
Vol 6 (1) ◽  
pp. 227-235 ◽  
Author(s):  
M M Haltiner ◽  
S T Smale ◽  
R Tjian
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Polymerase I ◽  
Upstream Region ◽  
Control Element ◽  
Rrna Gene ◽  
Start Site ◽  
Core Element ◽  
The Core ◽  
Polymerase I

A cell-free RNA polymerase I transcription system was used to evaluate the transcription efficiency of 21 linker scanning mutations that span the human rRNA gene promoter. Our analysis revealed the presence of two major control elements, designated the core and upstream elements, that affect the level of transcription initiation. The core element extends from -45 to +18 relative to the RNA start site, and transcription is severely affected (up to 100-fold) by linker scanning mutations in this region. Linker scanning and deletion mutations in the upstream element, located between nucleotides -156 and -107, cause a three- to fivefold reduction in transcription. Under certain reaction conditions, such as the presence of a high ratio of protein to template or supplementation of the reaction with partially purified protein fractions, sequences upstream of the core element can have an even greater effect (20- to 50-fold) on RNA polymerase I transcription. Primer extension analysis showed that RNA synthesized from all of these mutant templates is initiated at the correct in vivo start site. To examine the functional relationship between the core and the upstream region, mutant promoters were constructed that alter the orientation, distance, or multiplicity of these control elements relative to each other. The upstream control element appears to function in only one orientation, and its position relative to the core is constrained within a fairly narrow region. Moreover, multiple core elements in close proximity to each other have an inhibitory effect on transcription.

scholarly journals RNA polymerase I–specific subunits promote polymerase clustering to enhance the rRNA gene transcription cycle

2011 ◽  
Vol 192 (2) ◽  
pp. 277-293 ◽  
Author(s):  
Benjamin Albert ◽  
Isabelle Léger-Silvestre ◽  
Christophe Normand ◽  
Martin K. Ostermaier ◽  
Jorge Pérez-Fernández ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Loading Rate ◽  
Gene Copy Number ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Gene Copy ◽  
Rrna Gene ◽  
Pol I ◽  
Pol Iii ◽  
Polymerase I

RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I–specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.

scholarly journals Structural mechanism of ATP-independent transcription initiation by RNA polymerase I

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yan Han ◽  
Chunli Yan ◽  
Thi Hoang Duong Nguyen ◽  
Ashleigh J Jackobel ◽  
Ivaylo Ivanov ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Polymerase I ◽  
Independent Manner ◽  
Pol Ii ◽  
Structural Mechanism ◽  
The Core ◽  
Pol I ◽  
Upstream Promoter ◽  
Polymerase I

Transcription initiation by RNA Polymerase I (Pol I) depends on the Core Factor (CF) complex to recognize the upstream promoter and assemble into a Pre-Initiation Complex (PIC). Here, we solve a structure of Saccharomyces cerevisiae Pol I-CF-DNA to 3.8 Å resolution using single-particle cryo-electron microscopy. The structure reveals a bipartite architecture of Core Factor and its recognition of the promoter from −27 to −16. Core Factor’s intrinsic mobility correlates well with different conformational states of the Pol I cleft, in addition to the stabilization of either Rrn7 N-terminal domain near Pol I wall or the tandem winged helix domain of A49 at a partially overlapping location. Comparison of the three states in this study with the Pol II system suggests that a ratchet motion of the Core Factor-DNA sub-complex at upstream facilitates promoter melting in an ATP-independent manner, distinct from a DNA translocase actively threading the downstream DNA in the Pol II PIC.

Effect of intercalating agents on RNA polymerase I promoter selection in Xenopus laevis

1984 ◽  
Vol 4 (12) ◽  
pp. 2851-2857
Author(s):  
S C Pruitt ◽  
R H Reeder
Keyword(s):  
Xenopus Laevis ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Large Range ◽  
Gene Promoter ◽  
Rna Polymerase I ◽  
Polymerase I ◽  
Template Dna ◽  
Intercalating Agents ◽  
Transcription Signals

We have analyzed the effect of DNA intercalating agents on the transcription signals from two different Xenopus laevis RNA polymerase I promoters. The transcription signal from the promoter for the 7.5-kilobase rRNA precursor (the gene promoter) is unaffected over a large range of intercalating agent concentrations regardless of whether the template is injected plasmid DNA in oocytes, the amplified endogenous nucleoli of oocytes, or the endogenous chromosomes of cultured Xenopus kidney cells. The transcription signal from a closely related promoter located in the spacer DNA between genes (the spacer promoter) ranges between undetectable to equivalent to the gene promoter signal on different templates. The transcription signal from the spacer promoter is also differentially affected by intercalating agents relative to the gene promoter. Depending on the template, these agents can either increase or decrease the transcription signal from the spacer promoter. Fusions between the gene and spacer promoters demonstrate that intercalating agents affect transcription initiation. One explanation for these results is that the degree of supercoiling of the template DNA can differentially inhibit transcription from the spacer promoters. The different effects of intercalating agents on transcription from the spacer promoters of various templates could then be explained as differences in the degree of supercoiling present on these templates initially.

scholarly journals Regulation of Ribosomal RNA Production by RNA Polymerase I: Does Elongation Come First?

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Benjamin Albert ◽  
Jorge Perez-Fernandez ◽  
Isabelle Léger-Silvestre ◽  
Olivier Gadal
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Chromatin Structure ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Elongation Factors ◽  
Pol I ◽  
Polymerase I

Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35–47S) can be achieved by up to 150 RNA polymerase I (Pol I) enzymes simultaneously transcribing each rRNA gene. In this paper, we present recent advances made in understanding the regulatory mechanisms that control elongation. Built-in Pol I elongation factors, such as Rpa34/Rpa49 in budding yeast and PAF53/CAST in humans, are instrumental to the extremely high rate of rRNA production per gene. rRNA elongation mechanisms are intrinsically linked to chromatin structure and to the higher-order organization of the rRNA genes (rDNA). Factors such as Hmo1 in yeast and UBF1 in humans are key players in rDNA chromatin structure in vivo. Finally, elongation factors known to regulate messengers RNA production by RNA polymerase II are also involved in rRNA production and work cooperatively with Rpa49 in vivo.

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