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 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 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.

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.

Readthrough enhancement and promoter occlusion on the ribosomal genes of Xenopus laevis

1992 ◽  
Vol 70 (5) ◽  
pp. 324-331 ◽  
Author(s):  
Tom Moss ◽  
Anne-Marie Larose ◽  
Keith Mitchelson ◽  
Benoît Leblanc
Keyword(s):  
Xenopus Laevis ◽  
Rna Polymerase ◽  
Gene Promoter ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Relative Importance ◽  
Specific Mechanism ◽  
Enhancer Activity ◽  
Model Calculations ◽  
Polymerase I

An RNA polymerase I termination site is found just upstream of the ribosomal gene promoter in mammals and amphibia. It has been suggested that this termination site may actively enhance ribosomal transcription in a process known as readthrough enhancement or that it may simply prevent the disruption of initiation complexes or promoter occlusion. There is, however, a consensus of opinion that the terminator is important for efficient ribosomal transcription. Here we have quantitatively investigated the relative importance of readthrough enhancement and promoter occlusion on the transcription of the microinjected Xenopus laevis ribosomal gene. The results show that, in this system, promoter occlusion is limited and terminator mutations predominantly affect readthrough enhancement. The terminator is shown to be unnecessary for the enhancer activity of the rest of the ribosomal spacer. Model calculations suggest that readthrough enhancement could be explained by polymerase recycling and that it may be unnecessary to postulate a specific mechanism of polymerase handover.Key words: Xenopus, ribosomal, promoter, RNA polymerase I, rDNA.

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 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.

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