scholarly journals Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

1989 ◽  
Vol 9 (6) ◽  
pp. 2500-2512 ◽  
Author(s):  
K A Parker ◽  
U Bond
Keyword(s):  
Hela Cells ◽  
Transcription Termination ◽  
Control Level ◽  
Initiation Site ◽  
Transcription Unit ◽  
28S Rrna ◽  
Pol I ◽  
Nuclease Mapping ◽  
Polymerase I

Human rRNA precursor from normal or stressed HeLa cells were studied by S1 nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with [32P]PO4. Heating cells to 43 degrees C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 42 degrees C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol I initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identity with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 43 degrees C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs.

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription

1989 ◽  
Vol 9 (6) ◽  
pp. 2500-2512
Author(s):  
K A Parker ◽  
U Bond
Keyword(s):  
Hela Cells ◽  
Transcription Termination ◽  
Control Level ◽  
Initiation Site ◽  
Transcription Unit ◽  
28S Rrna ◽  
Pol I ◽  
Nuclease Mapping ◽  
Polymerase I

Human rRNA precursor from normal or stressed HeLa cells were studied by S1 nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with [32P]PO4. Heating cells to 43 degrees C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 42 degrees C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol I initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identity with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 43 degrees C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs.

scholarly journals Positive and Negative Autoregulation ofREB1 Transcription in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (7) ◽  
pp. 4368-4376 ◽  
Author(s):  
Kevin L.-C. Wang ◽  
Jonathan R. Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Initiation Site ◽  
Transcription Unit ◽  
Pol I ◽  
Activation Of Transcription

ABSTRACT Reb1p is a DNA binding protein of Saccharomyces cerevisiae that has been implicated in the activation of transcription by polymerase (Pol) II, in the termination of transcription by Pol I, and in the organization of nucleosomes. Studies of the transcriptional control of the REB1 gene have led us to identify three Reb1p binding sites in the 5′ region of the its gene, termed A, B, and C, at positions −110, −80, and +30 with respect to transcription initiation. In vitro, Reb1p binds to the three sites with the relative affinity of A ≥ C > B. Kinetic parameters suggest that when both A and C sites are present on the same DNA molecule, the C site may recruit Reb1p for the A site. In vivo the A and B sites each contribute to the transcription activity ofREB1 in roughly additive fashion. Mutation of both A and B sites abolishes transcription. On the other hand, the C site is a negative element, reducing transcription by 40%. In cells overexpressing Reb1p, the C site reduces transcription by more than 80%. This effect can be transposed to another transcription unit, demonstrating that the effect of Reb1p binding at the C site does not depend on interaction with upstream Reb1p molecules. Relocation of the C site to a position 105 bp downstream of the transcription initiation site abolishes its effect, suggesting that it does not act as a conventional attenuator of transcription. We conclude that binding of Reb1p at the C site hinders formation of the initiation complex. This arrangement of Reb1p binding sites provides a positive and negative mechanism to autoregulate the expression of REB1. Such an arrangement could serve to dampen the inevitable fluctuation in Rep1p levels caused by the intermittent presence of its mRNA within an individual cell.

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.

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.

Processing of eukaryotic pre-rRNA: the role of the transcribed spacers

1995 ◽  
Vol 73 (11-12) ◽  
pp. 789-801 ◽  
Author(s):  
Rob W. van Nues ◽  
Jaap Venema ◽  
Jeanette M. J. Rientjes ◽  
Anita Dirks-Mulder ◽  
Hendrik A. Raué
Keyword(s):  
Mutational Analysis ◽  
Structural Features ◽  
28S Rrna ◽  
Cis Acting ◽  
Processing Elements ◽  
Rrna Species ◽  
Polymerase I ◽  
Precursor Transcript

The 17–18S, 5.8S, and 25–28S rRNA species of eukaryotic cells are produced by a series of nucleolytic reactions that liberate the mature rRNAs from the large primary precursor transcript synthesized by RNA polymerase I. Whereas the order of the cleavage reactions has long been established, until recently little information was available on their molecular details, such as the nature of the proteins, including the nucleolytic enzymes, involved and the signals directing the processing machinery to the correct sites. This situation is now rapidly changing, in particular where yeast is concerned. The use of recently developed systems for in vivo mutational analysis of yeast rDNA has considerably enhanced our knowledge of cis-acting structural features within the pre-rRNA, in particular the transcribed spacer sequences, that are critical for correct and efficient removal of these spacers. The same systems also allow a link to be forged between trans-acting processing factors and these cis-acting elements. In this paper, we will focus predominantly on the nature and role of the cis-acting processing elements as identified in the transcribed spacer regions of Saccharomyces cerevisiae pre-rRNA.Key words: ribosome, processing, precursor rRNA, eukaryote, transcribed spacer.

scholarly journals The relationship between the activity of DNA-dependent RNA polymerase I and the rate of synthesis of rRNA in hepatoma cells in culture

1981 ◽  
Vol 194 (1) ◽  
pp. 43-51 ◽  
Author(s):  
E A Thompson ◽  
R H Keith ◽  
A H Cavanaugh ◽  
K M Wood
Keyword(s):  
Steady State ◽  
Rna Polymerase ◽  
Cell Lines ◽  
Rna Polymerase I ◽  
28S Rrna ◽  
Pol I ◽  
Cell Enzyme ◽  
Ribonucleoside Triphosphate ◽  
Polymerase I ◽  
The Relationship

Cell culture lines were established from the transplantable mouse hepatomas H6 and H129. Both cell lines had a doubling time about 30 h when maintained in medium containing 5% foetal bovine serum. H6 cells contained about 3-4 times more DNA-dependent RNA polymerase I (Pol I; ribonucleoside triphosphate–RNA nucleotidyltransferase, EC 2.7.7.6) than did H129 cells. Moreover, the H6-cell enzyme was more heat-labile than that from H129 cells. Steady-state contents of 28S rRNA were measured in both cell lines. Exponentially growing cultures of H6 cells contained about 6.5pg of 28S rRNA/cell, and similar cultures of H129 cells contained about 5.8pg/cell. Stationary cultures of both cell lines contained about 2pg of 28S rRNA/cell. By two different techniques, the half-time for turnover of 28S rRNA was estimated to be 16-17h for both H6 and H129 cells. Knowing the turnover rate and the steady-state concentration, one may calculate that both H6 and H129 cells synthesize 28S rRNA at a rate of about 0.25 pg/h per cell. The amount of template-bound Pol I activity was similar in nuclei isolated from H6 and H129 cell cultures. These data indicate that, although H6 cells contained 3-4 times more Pol I than did H129 cells, both cell lines synthesized rRNA at about the same rate.

scholarly journals RNA Polymerase I-Promoted HIS4Expression Yields Uncapped, Polyadenylated mRNA That Is Unstable and Inefficiently Translated in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (2) ◽  
pp. 665-675 ◽  
Author(s):  
Hsiu-Jung Lo ◽  
Han-Kuei Huang ◽  
Thomas F. Donahue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Mutant Form ◽  
Wild Type ◽  
Pol I ◽  
Chromosome Iii ◽  
Polymerase I

ABSTRACT The HIS4 gene in Saccharomyces cerevisiaewas put under the transcriptional control of RNA polymerase I to determine the in vivo consequences on mRNA processing and gene expression. This gene, referred to as rhis4, was substituted for the normal HIS4 gene on chromosome III. Therhis4 gene transcribes two mRNAs, of which each initiates at the polymerase (pol) I transcription initiation site. One transcript, rhis4s, is similar in size to the wild-typeHIS4 mRNA. Its 3′ end maps to the HIS4 3′ noncoding region, and it is polyadenylated. The second transcript,rhis4l, is bicistronic. It encodes the HIS4coding region and a second open reading frame, YCL184, that is located downstream of the HIS4 gene and is predicted to be transcribed in the same direction as HIS4 on chromosome III. The 3′ end of rhis4l maps to the predicted 3′ end of the YCL184 gene and is also polyadenylated. Based on in vivo labeling experiments, the rhis4 gene appears to be more actively transcribed than the wild-type HIS4 gene despite the near equivalence of the steady-state levels of mRNAs produced from each gene. This finding indicated that rhis4mRNAs are rapidly degraded, presumably due to the lack of a cap structure at the 5′ end of the mRNA. Consistent with this interpretation, a mutant form of XRN1, which encodes a 5′-3′ exonuclease, was identified as an extragenic suppressor that increases the half-life of rhis4 mRNA, leading to a 10-fold increase in steady-state mRNA levels compared to the wild-typeHIS4 mRNA level. This increase is dependent on pol I transcription. Immunoprecipitation by anticap antiserum suggests that the majority of rhis4 mRNA produced is capless. In addition, we quantitated the level of His4 protein in a rhis4 xrn1Δ genetic background. This analysis indicates that capless mRNA is translated at less than 10% of the level of translation of capped HIS4 mRNA. Our data indicate that polyadenylation of mRNA in yeast occurs despite HIS4 being transcribed by RNA polymerase I, and the 5′ cap confers stability to mRNA and affords the ability of mRNA to be translated efficiently in vivo.

scholarly journals CX-5461 Treatment Leads to Cytosolic DNA-Mediated STING Activation in Ovarian Cancer

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5056
Author(s):  
Robert Cornelison ◽  
Kuntal Biswas ◽  
Danielle C. Llaneza ◽  
Alexandra R. Harris ◽  
Nisha G. Sosale ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Time Frame ◽  
Interferon Response ◽  
Type I ◽  
Pol I ◽  
Downstream Effects ◽  
Immune Pathway ◽  
Polymerase I

Epithelial ovarian cancer (EOC) is the deadliest of the gynecologic malignancies, with an overall survival rate of <30%. Recent research has suggested that targeting RNA polymerase I (POL I) with small-molecule inhibitors may be a viable therapeutic approach to combating EOC, even when chemoresistance is present. CX-5461 is one of the most promising POL I inhibitors currently being investigated, and previous reports have shown that CX-5461 treatment induces DNA damage response (DDR) through ATM/ATR kinase. Investigation into downstream effects of CX-5461 led us to uncovering a previously unreported phenotype. Treatment with CX-5461 induces a rapid accumulation of cytosolic DNA. This accumulation leads to transcriptional upregulation of ‘STimulator of Interferon Genes’ (STING) in the same time frame, phosphorylation of IRF3, and activation of type I interferon response both in vitro and in vivo. This activation is mediated and dependent on cyclic GMP–AMP synthase (cGAS). Here, we show THAT CX-5461 leads to an accumulation of cytosolic dsDNA and thereby activates the cGAS–STING–TBK1–IRF3 innate immune pathway, which induces type I IFN. CX-5461 treatment-mediated immune activation may be a powerful mechanism of action to exploit, leading to novel drug combinations with a chance of increasing immunotherapy efficacy, possibly with some cancer specificity limiting deleterious toxicities.

Dynamics of the RNA polymerase I TFIIF/TFIIE-like subcomplex: a mini-review

2020 ◽  
Vol 48 (5) ◽  
pp. 1917-1927
Author(s):  
Bruce A. Knutson ◽  
Rachel McNamar ◽  
Lawrence I. Rothblum
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Genetic Disorders ◽  
Rna Polymerase I ◽  
28S Rrna ◽  
Pol I ◽  
Transcription Process ◽  
Polymerase I ◽  
Linker Domain ◽  
Dynamic Interplay

RNA polymerase I (Pol I) is the most specialized eukaryotic Pol. It is only responsible for the synthesis of pre-ribosomal RNA (rRNA), the precursor of 18S, 5.8S and 28S rRNA, the most abundant cellular RNA types. Aberrant Pol I transcription is observed in a wide variety of cancers and its down-regulation is associated with several genetic disorders. The regulation and mechanism of Pol I transcription is increasing in clarity given the numerous high-resolution Pol I structures that have helped bridge seminal genetic and biochemical findings in the field. Here, we review the multifunctional roles of an important TFIIF- and TFIIE-like subcomplex composed of the Pol I subunits A34.5 and A49 in yeast, and PAF49 and PAF53 in mammals. Recent analyses have revealed a dynamic interplay between this subcomplex at nearly every step of the Pol I transcription cycle in addition to new roles in chromatin traversal and the existence of a new helix-turn-helix (HTH) within the A49/PAF53 linker domain that expands its dynamic functions during the Pol I transcription process.

Transcription of the mouse ribosomal spacer region

1984 ◽  
Vol 4 (5) ◽  
pp. 822-828
Author(s):  
K M Wood ◽  
L H Bowman ◽  
E A Thompson
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Blot Hybridization ◽  
Rna Polymerase I ◽  
Dot Blot ◽  
Intact Cells ◽  
Nuclease Mapping ◽  
Polymerase I

This paper describes experiments designed to test the hypothesis that DNA sequences upstream from the mouse rRNA promoter are transcribed in vivo or in vitro. Plasmid pB28 contains a SalI restriction fragment that extends from -169 to -1,894 base pairs, with respect to the origin of transcription of pre-rRNA. Labeled RNA synthesized in intact cells does not hybridize to this region. Neither S1 nuclease mapping nor RNA dot blot hybridization revealed the presence of sequences complementary to this region. Transcriptional studies carried out in vitro indicated that this region is not transcribed under conditions that are optimal for utilization of the authentic rRNA promoter. Moreover, this region does not appear to form stable transcription complexes with RNA polymerase I transcription components. These data indicate that the mouse rDNA repeating unit differs from those of Xenopus spp. and Drosophila melanogaster in that reduplicated RNA polymerase I promoters are not found in the mouse rDNA spacer region.

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