Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene

1987 ◽  
Vol 7 (1) ◽  
pp. 314-325
Author(s):  
C A Harrington ◽  
D M Chikaraishi
Keyword(s):  
Ribosomal Dna ◽  
Transcriptional Activity ◽  
Tandem Repeats ◽  
Initiation Site ◽  
Rrna Synthesis ◽  
Rna Transcripts ◽  
Pair Sequence ◽  
Polymerase I

The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

scholarly journals Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene.

1987 ◽  
Vol 7 (1) ◽  
pp. 314-325 ◽  
Author(s):  
C A Harrington ◽  
D M Chikaraishi
Keyword(s):  
Ribosomal Dna ◽  
Transcriptional Activity ◽  
Tandem Repeats ◽  
Initiation Site ◽  
Rrna Synthesis ◽  
Rna Transcripts ◽  
Pair Sequence ◽  
Polymerase I

The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

scholarly journals The Xenopus ribosomal DNA 60- and 81-base-pair repeats are position-dependent enhancers that function at the establishment of the preinitiation complex: analysis in vivo and in an enhancer-responsive in vitro system.

1989 ◽  
Vol 9 (11) ◽  
pp. 5093-5104 ◽  
Author(s):  
L K Pape ◽  
J J Windle ◽  
E B Mougey ◽  
B Sollner-Webb
Keyword(s):  
Ribosomal Dna ◽  
Base Pair ◽  
Complex Analysis ◽  
Initiation Site ◽  
In Vitro System ◽  
Sequential Addition ◽  
Polymerase I ◽  
In Cis

Although it is generally believed that the 60- and 81-base-pair (60/81-bp) repeats of the Xenopus laevis ribosomal DNA (rDNA) spacer are position-independent transcriptional enhancers, this has not been shown directly. We have now developed a critical assay which proves that the 60/81-bp repeats do, in fact, stimulate transcription from promoters in cis and that they function in both orientations and when up to 1 kilobase pair from the initiation site. However, contrary to the widely accepted view, these elements are found to be highly position dependent, for they have no net effect when downstream of the initiation site within the transcribed region and they behave as transcriptional silencers of promoters in cis when moved greater than 2 kilobase pairs upstream of the initiation site. The 60/81-bp elements therefore are position-dependent 5' enhancers. We also found that this rDNA enhancer was polymerase I specific and that it was composed of duplicated, individually functional elements. Finally, we report an in vitro system that reproduces both cis enhancement and trans competition by the 60/81-bp repeats. Sequential-addition studies in this system demonstrated that the rDNA enhancer functions in trans at or before establishment of the stable transcription complex, not subsequently at each round of transcription.

The Xenopus ribosomal DNA 60- and 81-base-pair repeats are position-dependent enhancers that function at the establishment of the preinitiation complex: analysis in vivo and in an enhancer-responsive in vitro system

1989 ◽  
Vol 9 (11) ◽  
pp. 5093-5104
Author(s):  
L K Pape ◽  
J J Windle ◽  
E B Mougey ◽  
B Sollner-Webb
Keyword(s):  
Ribosomal Dna ◽  
Base Pair ◽  
Complex Analysis ◽  
Initiation Site ◽  
In Vitro System ◽  
Sequential Addition ◽  
Polymerase I ◽  
In Cis

Although it is generally believed that the 60- and 81-base-pair (60/81-bp) repeats of the Xenopus laevis ribosomal DNA (rDNA) spacer are position-independent transcriptional enhancers, this has not been shown directly. We have now developed a critical assay which proves that the 60/81-bp repeats do, in fact, stimulate transcription from promoters in cis and that they function in both orientations and when up to 1 kilobase pair from the initiation site. However, contrary to the widely accepted view, these elements are found to be highly position dependent, for they have no net effect when downstream of the initiation site within the transcribed region and they behave as transcriptional silencers of promoters in cis when moved greater than 2 kilobase pairs upstream of the initiation site. The 60/81-bp elements therefore are position-dependent 5' enhancers. We also found that this rDNA enhancer was polymerase I specific and that it was composed of duplicated, individually functional elements. Finally, we report an in vitro system that reproduces both cis enhancement and trans competition by the 60/81-bp repeats. Sequential-addition studies in this system demonstrated that the rDNA enhancer functions in trans at or before establishment of the stable transcription complex, not subsequently at each round of transcription.

scholarly journals A complex control region of the mouse rRNA gene directs accurate initiation by RNA polymerase I.

1985 ◽  
Vol 5 (3) ◽  
pp. 554-562 ◽  
Author(s):  
K G Miller ◽  
J Tower ◽  
B Sollner-Webb
Keyword(s):  
Initiation Site ◽  
Rrna Gene ◽  
Sufficient Information ◽  
Reaction Conditions ◽  
Cell Extracts ◽  
S 100 ◽  
Initiation Reaction ◽  
Polymerase I

To determine the size and location of the mouse rDNA promoter, we constructed systematic series of deletion mutants approaching the initiation site from the 5' and 3' directions. These templates were transcribed in vitro under various conditions with S-100 and whole-cell extracts. Surprisingly, the size of the rDNA region that determines the level of transcription differed markedly, depending on the reaction conditions. In both kinds of cell extracts, the apparent 5' border of the promoter was at residue ca. -27 under optimal transcription conditions, but as reaction conditions became less favorable, the 5' border moved progressively out to residues -35, -39, and -45. The complete promoter, however, extends considerably further, for under other nonoptimal conditions, we observed major effects of promoter domains extending in the 5' direction to positions ca. -100 and -140. In contrast, the apparent 3' border of the mouse rDNA promoter was at residue ca. +9 under all conditions examined. We also show that the subcloned rDNA region from -39 to +9 contains sufficient information to initiate accurately and that the region between +2 and +9 can influence the specificity of initiation. These data indicate that, although the polymerase I transcription factors recognize and accurately initiate with only the sequences downstream of residue -40, sequences extending out to residue -140 greatly favor the initiation reaction; presumably, this entire region is involved in rRNA transcription in vivo.

scholarly journals Interaction of TATA-Binding Protein with Upstream Activation Factor Is Required for Activated Transcription of Ribosomal DNA by RNA Polymerase I in Saccharomyces cerevisiae In Vivo

1998 ◽  
Vol 18 (7) ◽  
pp. 3752-3761 ◽  
Author(s):  
Joan S. Steffan ◽  
Daniel A. Keys ◽  
Loan Vu ◽  
Masayasu Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hybrid System ◽  
Ribosomal Dna ◽  
Tata Binding Protein ◽  
Rna Polymerase I ◽  
Activation Factor ◽  
Polymerase I ◽  
Two Hybrid

ABSTRACT Previous in vitro studies have shown that initiation of transcription of ribosomal DNA (rDNA) in the yeast Saccharomyces cerevisiae involves an interaction of upstream activation factor (UAF) with the upstream element of the promoter, forming a stable UAF-template complex; together with TATA-binding protein (TBP), UAF then recruits an essential factor, core factor (CF), to the promoter, forming a stable preinitiation complex. TBP interacts with both UAF and CF in vitro. In addition, a subunit of UAF, Rrn9p, interacts with TBP in vitro and in the two-hybrid system, suggesting the possible importance of this interaction for UAF function. Using the yeast two-hybrid system, we have identified three mutations inRRN9 that abolish the interaction of Rrn9p with TBP without affecting its interaction with Rrn10p, another subunit of UAF. Yeast cells containing any one of these individual mutations,L110S, L269P, or L274Q, did not show any growth defects. However, cells containing a combination ofL110S with one of the other two mutations showed a temperature-sensitive phenotype, and this phenotype was suppressed by fusing the mutant genes to SPT15, which encodes TBP. In addition, another mutation (F186S), which disrupts both Rrn9p-TBP and Rrn9p-Rrn10p interactions in the two-hybrid system, abolished UAF function in vivo, and this mutational defect was suppressed by fusion of the mutant gene to SPT15 combined with overexpression of Rrn10p. These experiments demonstrate that the interaction of UAF with TBP, which is presumably achieved by the interaction of Rrn9p with TBP, is indeed important for high-level transcription of rDNA by RNA polymerase I in vivo.

scholarly journals Transcription of Multiple Yeast Ribosomal DNA Genes Requires Targeting of UAF to the Promoter by Uaf30

2008 ◽  
Vol 28 (21) ◽  
pp. 6709-6719 ◽  
Author(s):  
Robert D. Hontz ◽  
Sarah L. French ◽  
Melanie L. Oakes ◽  
Prasad Tongaonkar ◽  
Masayasu Nomura ◽  
...  
Keyword(s):  
Ribosomal Dna ◽  
Rrna Synthesis ◽  
Synthesis Rate ◽  
Wild Type ◽  
Rdna Transcription ◽  
Rdna Genes ◽  
Pol I ◽  
Polymerase I ◽  
I Cells

ABSTRACT Upstream activating factor (UAF) is a multisubunit complex that functions in the activation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I). Cells lacking the Uaf30 subunit of UAF reduce the rRNA synthesis rate by ∼70% compared to wild-type cells and produce rRNA using both Pol I and Pol II. Miller chromatin spreads demonstrated that even though there is an overall reduction in rRNA synthesis in uaf30 mutants, the active rDNA genes in such strains are overloaded with polymerases. This phenotype was specific to defects in Uaf30, as mutations in other UAF subunits resulted in a complete absence of rDNA genes with high or even modest Pol densities. The lack of Uaf30 prevented UAF from efficiently binding to the rDNA promoter in vivo, leading to an inability to activate a large number of rDNA genes. The relatively few genes that did become activated were highly transcribed, apparently to compensate for the reduced rRNA synthesis capacity. The results show that Uaf30p is a key targeting factor for the UAF complex that facilitates activation of a large proportion of rDNA genes in the tandem array.

scholarly journals Casein Kinase 2 Associates with Initiation-Competent RNA Polymerase I and Has Multiple Roles in Ribosomal DNA Transcription

2006 ◽  
Vol 26 (16) ◽  
pp. 5957-5968 ◽  
Author(s):  
Tatiana B. Panova ◽  
Kostya I. Panov ◽  
Jackie Russell ◽  
Joost C. B. M. Zomerdijk
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Rna Polymerase I ◽  
Positive Effects ◽  
Pol I ◽  
Polymerase I

ABSTRACT Mammalian RNA polymerase I (Pol I) complexes contain a number of associated factors, some with undefined regulatory roles in transcription. We demonstrate that casein kinase 2 (CK2) in human cells is associated specifically only with the initiation-competent Pol Iβ isoform and not with Pol Iα. Chromatin immunoprecipitation analysis places CK2 at the ribosomal DNA (rDNA) promoter in vivo. Pol Iβ-associated CK2 can phosphorylate topoisomerase IIα in Pol Iβ, activator upstream binding factor (UBF), and selectivity factor 1 (SL1) subunit TAFI110. A potent and selective CK2 inhibitor, 3,8-dibromo-7-hydroxy-4-methylchromen-2-one, limits in vitro transcription to a single round, suggesting a role for CK2 in reinitiation. Phosphorylation of UBF by CK2 increases SL1-dependent stabilization of UBF at the rDNA promoter, providing a molecular mechanism for the stimulatory effect of CK2 on UBF activation of transcription. These positive effects of CK2 in Pol I transcription contrast to that wrought by CK2 phosphorylation of TAFI110, which prevents SL1 binding to rDNA, thereby abrogating the ability of SL1 to nucleate preinitiation complex (PIC) formation. Thus, CK2 has the potential to regulate Pol I transcription at multiple levels, in PIC formation, activation, and reinitiation of transcription.

scholarly journals Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

2018 ◽  
Author(s):  
Tommy Darrière ◽  
Michael Pilsl ◽  
Marie-Kerguelen Sarthou ◽  
Adrien Chauvier ◽  
Titouan Genty ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Growth Defect ◽  
Pol I ◽  
Polymerase I ◽  
Extragenic Suppressors

AbstractMost transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that wild-type RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.Author summaryThe nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.

A complex control region of the mouse rRNA gene directs accurate initiation by RNA polymerase I

1985 ◽  
Vol 5 (3) ◽  
pp. 554-562
Author(s):  
K G Miller ◽  
J Tower ◽  
B Sollner-Webb
Keyword(s):  
Initiation Site ◽  
Rrna Gene ◽  
Sufficient Information ◽  
Reaction Conditions ◽  
Cell Extracts ◽  
S 100 ◽  
Initiation Reaction ◽  
Polymerase I

To determine the size and location of the mouse rDNA promoter, we constructed systematic series of deletion mutants approaching the initiation site from the 5' and 3' directions. These templates were transcribed in vitro under various conditions with S-100 and whole-cell extracts. Surprisingly, the size of the rDNA region that determines the level of transcription differed markedly, depending on the reaction conditions. In both kinds of cell extracts, the apparent 5' border of the promoter was at residue ca. -27 under optimal transcription conditions, but as reaction conditions became less favorable, the 5' border moved progressively out to residues -35, -39, and -45. The complete promoter, however, extends considerably further, for under other nonoptimal conditions, we observed major effects of promoter domains extending in the 5' direction to positions ca. -100 and -140. In contrast, the apparent 3' border of the mouse rDNA promoter was at residue ca. +9 under all conditions examined. We also show that the subcloned rDNA region from -39 to +9 contains sufficient information to initiate accurately and that the region between +2 and +9 can influence the specificity of initiation. These data indicate that, although the polymerase I transcription factors recognize and accurately initiate with only the sequences downstream of residue -40, sequences extending out to residue -140 greatly favor the initiation reaction; presumably, this entire region is involved in rRNA transcription in vivo.

The mouse ribosomal DNA promoter has more stringent requirements in vivo than in vitro

1990 ◽  
Vol 10 (9) ◽  
pp. 4970-4973
Author(s):  
S L Henderson ◽  
B Sollner-Webb
Keyword(s):  
Ribosomal Dna ◽  
Promoter Analysis ◽  
Core Region ◽  
Transcriptional Interference ◽  
Intact Cells ◽  
Dna Templates ◽  
The Core ◽  
Polymerase I

Using mouse ribosomal DNA templates bearing polymerase I terminators to prevent transcriptional interference (S. L. Henderson, K. Ryan, and B. Sollner-Webb, Genes Dev. 3:212-223, 1989) and facilitate promoter analysis in intact cells, we demonstrate that a -140 promoter domain (as well as the core region) is essential for appreciable levels of initiation in vivo. This in vivo polymerase I promoter can also be detected in vitro but only under very stringent conditions.

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