Widespread Use of TATA Elements in the Core Promoters for RNA Polymerases III, II, and I in Fission Yeast

ABSTRACT In addition to directing transcription initiation, core promoters integrate input from distal regulatory elements. Except for rare exceptions, it has been generally found that eukaryotic tRNA and rRNA genes do not contain TATA promoter elements and instead use protein-protein interactions to bring the TATA-binding protein (TBP), to the core promoter. Genomewide analysis revealed TATA elements in the core promoters of tRNA and 5S rRNA (Pol III), U1 to U5 snRNA (Pol II), and 37S rRNA (Pol I) genes in Schizosaccharomyces pombe. Using tRNA-dependent suppression and other in vivo assays, as well as in vitro transcription, we demonstrated an obligatory requirement for upstream TATA elements for tRNA and 5S rRNA expression in S. pombe. The Pol III initiation factor Brf is found in complexes with TFIIIC and Pol III in S. pombe, while TBP is not, consistent with independent recruitment of TBP by TATA. Template commitment assays are consistent with this and confirm that the mechanisms of transcription complex assembly and initiation by Pol III in S. pombe differ substantially from those in other model organisms. The results were extended to large-rRNA synthesis, as mutation of the TATA element in the Pol I promoter also abolishes rRNA expression in fission yeast. A survey of other organisms' genomes reveals that a substantial number of eukaryotes may use widespread TATAs for transcription. These results indicate the presence of TATA-unified transcription systems in contemporary eukaryotes and provide insight into the residual need for TBP by all three Pols in other eukaryotes despite a lack of TATA elements in their promoters.

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Harnessing the Nucleolar DNA Damage Response in Cancer Therapy

Genes ◽

10.3390/genes12081156 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1156

Author(s):

Jiachen Xuan ◽

Kezia Gitareja ◽

Natalie Brajanovski ◽

Elaine Sanij

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Cancer Therapy ◽

Dna Damage Response ◽

Rrna Genes ◽

Rrna Synthesis ◽

Pol I ◽

Clinical Investigations ◽

Damage Response ◽

Synthesis And Processing

The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. They serve as the site of rRNA synthesis and processing, and ribosome assembly. There are 400–600 copies of rRNA genes (rDNA) in human cells and their highly repetitive and transcribed nature poses a challenge for DNA repair and replication machineries. It is only in the last 7 years that the DNA damage response and processes of DNA repair at the rDNA repeats have been recognized to be unique and distinct from the classic response to DNA damage in the nucleoplasm. In the last decade, the nucleolus has also emerged as a central hub for coordinating responses to stress via sequestering tumor suppressors, DNA repair and cell cycle factors until they are required for their functional role in the nucleoplasm. In this review, we focus on features of the rDNA repeats that make them highly vulnerable to DNA damage and the mechanisms by which rDNA damage is repaired. We highlight the molecular consequences of rDNA damage including activation of the nucleolar DNA damage response, which is emerging as a unique response that can be exploited in anti-cancer therapy. In this review, we focus on CX-5461, a novel inhibitor of Pol I transcription that induces the nucleolar DNA damage response and is showing increasing promise in clinical investigations.

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Identification and characterization of cis-regulatory elements for photoreceptor type-specific transcription in zebrafish

10.1101/683284 ◽

2019 ◽

Author(s):

Wei Fang ◽

Yi Wen ◽

Xiangyun Wei

Keyword(s):

Core Promoter ◽

Regulatory Elements ◽

Specific Gene ◽

Protein Coding ◽

Core Promoters ◽

Protein Coding Genes ◽

The Core ◽

Cell Type Specific ◽

Identification And Characterization

AbstractTissue-specific or cell type-specific transcription of protein-coding genes is controlled by both trans-regulatory elements (TREs) and cis-regulatory elements (CREs). However, it is challenging to identify TREs and CREs, which are unknown for most genes. Here, we describe a protocol for identifying two types of transcription-activating CREs—core promoters and enhancers—of zebrafish photoreceptor type-specific genes. This protocol is composed of three phases: bioinformatic prediction, experimental validation, and characterization of the CREs. To better illustrate the principles and logic of this protocol, we exemplify it with the discovery of the core promoter and enhancer of the mpp5b apical polarity gene (also known as ponli), whose red, green, and blue (RGB) cone-specific transcription requires its enhancer, a member of the rainbow enhancer family. While exemplified with an RGB cone-specific gene, this protocol is general and can be used to identify the core promoters and enhancers of other protein-coding genes.

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Long Tandem Arrays of Cassandra Retroelements and Their Role in Genome Dynamics in Plants

International Journal of Molecular Sciences ◽

10.3390/ijms21082931 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2931 ◽

Cited By ~ 5

Author(s):

Ruslan Kalendar ◽

Olga Raskina ◽

Alexander Belyayev ◽

Alan H. Schulman

Keyword(s):

Copy Number ◽

5S Rdna ◽

5S Rrna ◽

Rrna Genes ◽

Gene Copy ◽

Rrna Gene ◽

Long Terminal Repeats ◽

5S Rrna Genes ◽

Pol Iii ◽

Tandem Arrays

Retrotransposable elements are widely distributed and diverse in eukaryotes. Their copy number increases through reverse-transcription-mediated propagation, while they can be lost through recombinational processes, generating genomic rearrangements. We previously identified extensive structurally uniform retrotransposon groups in which no member contains the gag, pol, or env internal domains. Because of the lack of protein-coding capacity, these groups are non-autonomous in replication, even if transcriptionally active. The Cassandra element belongs to the non-autonomous group called terminal-repeat retrotransposons in miniature (TRIM). It carries 5S RNA sequences with conserved RNA polymerase (pol) III promoters and terminators in its long terminal repeats (LTRs). Here, we identified multiple extended tandem arrays of Cassandra retrotransposons within different plant species, including ferns. At least 12 copies of repeated LTRs (as the tandem unit) and internal domain (as a spacer), giving a pattern that resembles the cellular 5S rRNA genes, were identified. A cytogenetic analysis revealed the specific chromosomal pattern of the Cassandra retrotransposon with prominent clustering at and around 5S rDNA loci. The secondary structure of the Cassandra retroelement RNA is predicted to form super-loops, in which the two LTRs are complementary to each other and can initiate local recombination, leading to the tandem arrays of Cassandra elements. The array structures are conserved for Cassandra retroelements of different species. We speculate that recombination events similar to those of 5S rRNA genes may explain the wide variation in Cassandra copy number. Likewise, the organization of 5S rRNA gene sequences is very variable in flowering plants; part of what is taken for 5S gene copy variation may be variation in Cassandra number. The role of the Cassandra 5S sequences remains to be established.

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Spt6 Is Essential for rRNA Synthesis by RNA Polymerase I

Molecular and Cellular Biology ◽

10.1128/mcb.01499-14 ◽

2015 ◽

Vol 35 (13) ◽

pp. 2321-2331 ◽

Cited By ~ 4

Author(s):

Krysta L. Engel ◽

Sarah L. French ◽

Olga V. Viktorovskaya ◽

Ann L. Beyer ◽

David A. Schneider

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Initiation Factor ◽

Rrna Synthesis ◽

Temperature Sensitive ◽

Protein Levels ◽

Pol I ◽

Low Levels ◽

Sensitive Allele ◽

Polymerase I

Spt6 (suppressor ofTy6) has many roles in transcription initiation and elongation by RNA polymerase (Pol) II. These effects are mediated through interactions with histones, transcription factors, and the RNA polymerase. Two lines of evidence suggest that Spt6 also plays a role in rRNA synthesis. First, Spt6 physically associates with a Pol I subunit (Rpa43). Second, Spt6 interacts physically and genetically with Spt4/5, which directly affects Pol I transcription. Utilizing a temperature-sensitive allele,spt6-1004, we show that Spt6 is essential for Pol I occupancy of the ribosomal DNA (rDNA) and rRNA synthesis. Our data demonstrate that protein levels of an essential Pol I initiation factor, Rrn3, are reduced when Spt6 is inactivated, leading to low levels of Pol I-Rrn3 complex. Overexpression ofRRN3rescues Pol I-Rrn3 complex formation; however, rRNA synthesis is not restored. These data suggest that Spt6 is involved in either recruiting the Pol I-Rrn3 complex to the rDNA or stabilizing the preinitiation complex. The findings presented here identify an unexpected, essential role for Spt6 in synthesis of rRNA.

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In Exponentially Growing Saccharomyces cerevisiae Cells, rRNA Synthesis Is Determined by the Summed RNA Polymerase I Loading Rate Rather than by the Number of Active Genes

Molecular and Cellular Biology ◽

10.1128/mcb.23.5.1558-1568.2003 ◽

2003 ◽

Vol 23 (5) ◽

pp. 1558-1568 ◽

Cited By ~ 197

Author(s):

Sarah L. French ◽

Yvonne N. Osheim ◽

Francesco Cioci ◽

Masayasu Nomura ◽

Ann L. Beyer

Keyword(s):

Saccharomyces Cerevisiae ◽

Exponential Growth ◽

Rrna Genes ◽

Rrna Synthesis ◽

Rrna Gene ◽

Transcription Rate ◽

Initiation Rate ◽

Pol I ◽

Genes Encoding ◽

Active Genes

ABSTRACT Genes encoding rRNA are multicopy and thus could be regulated by changing the number of active genes or by changing the transcription rate per gene. We tested the hypothesis that the number of open genes is limiting rRNA synthesis by using an electron microscopy method that allows direct counting of the number of active genes per nucleolus and the number of polymerases per active gene. Two strains of Saccharomyces cerevisiae were analyzed during exponential growth: a control strain with a typical number of rRNA genes (∼143 in this case) and a strain in which the rRNA gene number was reduced to ∼42 but which grows as well as controls. In control strains, somewhat more than half of the genes were active and the mean number of polymerases/gene was ∼50 ± 20. In the 42-copy strain, all rRNA genes were active with a mean number of 100 ± 29 polymerases/gene. Thus, an equivalent number of polymerases was active per nucleolus in the two strains, though the number of active genes varied by twofold, showing that overall initiation rate, and not the number of active genes, determines rRNA transcription rate during exponential growth in yeast. Results also allow an estimate of elongation rate of ∼60 nucleotides/s for yeast Pol I and a reinitiation rate of less than 1 s on the most heavily transcribed genes.

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In vivo regulation of rRNA transcription occurs rapidly in nondividing and dividing Drosophila cells in response to a phorbol ester and serum.

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.928 ◽

1993 ◽

Vol 13 (2) ◽

pp. 928-933 ◽

Cited By ~ 9

Author(s):

S M Vallett ◽

M Brudnak ◽

M Pellegrini ◽

H W Weber

Keyword(s):

Phorbol Ester ◽

Growth Regulation ◽

Rrna Genes ◽

Rrna Synthesis ◽

Rrna Gene ◽

Rdna Transcription ◽

Transcription System ◽

Pol I ◽

Drosophila Cells

The synthesis of ribosomes is an essential cellular process which requires the transcription of the rRNA genes by RNA polymerase I (Pol I). The regulation of rRNA synthesis is known to be coupled to growth regulation. In nongrowing, slowly growing, and rapidly growing Drosophila cells, exposure to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) increases the synthesis of precursor and mature rRNAs. Using nuclear run-on assays, we show that TPA enhances transcription of the rRNA genes. These results suggest that TPA regulates expression of RNA genes transcribed by Pol I, irrespective of the growth state of the cells. In slowly dividing Drosophila cells, increasing the serum concentration rapidly alters the accumulation of rRNA by enhancing rDNA transcription within 1 h. Thus, TPA and serum are each able to rapidly regulate rRNA gene expression in Drosophila cells. These results indicate that the RNA Pol I transcription system can be regulated by agents which have previously been shown to effect specific genes transcribed by the RNA Pol II system.

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Rapamycin Induces the G0 Program of Transcriptional Repression in Yeast by Interfering with the TOR Signaling Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.18.8.4463 ◽

1998 ◽

Vol 18 (8) ◽

pp. 4463-4470 ◽

Cited By ~ 153

Author(s):

Dean Zaragoza ◽

Ataollah Ghavidel ◽

Joseph Heitman ◽

Michael C. Schultz

Keyword(s):

Signaling Pathway ◽

Transcriptional Repression ◽

Transcription Initiation ◽

Cellular Proliferation ◽

Yeast Cells ◽

Initiation Factor ◽

Independent Effect ◽

Tor Signaling ◽

Pol I ◽

Pol Iii

ABSTRACT The macrolide antibiotic rapamycin inhibits cellular proliferation by interfering with the highly conserved TOR (for target of rapamycin) signaling pathway. Growth arrest of budding yeast cells treated with rapamycin is followed by the program of molecular events that characterizes entry into G0 (stationary phase), including the induction of polymerase (Pol) II genes typically expressed only in G0. Normally, progression into G0 is characterized by transcriptional repression of the Pol I and III genes. Here, we show that rapamycin treatment also causes the transcriptional repression of Pol I and III genes. The down-regulation of Pol III transcription is TOR dependent. While it coincides with translational repression by rapamycin, transcriptional repression is due in part to a translation-independent effect that is evident in extracts from a conditional tor2 mutant. Biochemical experiments reveal that RNA Pol III and probably transcription initiation factor TFIIIB are targets of repression by rapamycin. In view of previous evidence that TFIIIB and Pol III are inhibited when protein phosphatase 2A (PP2A) function is impaired, and that PP2A is a component of the TOR pathway, our results suggest that TOR signaling regulates Pol I and Pol III transcription in response to nutrient growth signals.

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Role of Histone H1 as an Architectural Determinant of Chromatin Structure and as a Specific Repressor of Transcription onXenopus Oocyte 5S rRNA Genes

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.3668 ◽

1998 ◽

Vol 18 (7) ◽

pp. 3668-3680 ◽

Cited By ~ 62

Author(s):

Takashi Sera ◽

Alan P. Wolffe

Keyword(s):

Chromatin Structure ◽

Regulatory Elements ◽

Histone H1 ◽

5S Rrna ◽

Rrna Genes ◽

Rrna Gene ◽

Nucleosome Core ◽

5S Rrna Gene ◽

5S Rrna Genes

ABSTRACT We explore the role of histone H1 as a DNA sequence-dependent architectural determinant of chromatin structure and of transcriptional activity in chromatin. The Xenopus laevis oocyte- and somatic-type 5S rRNA genes are differentially transcribed in embryonic chromosomes in vivo depending on the incorporation of somatic histone H1 into chromatin. We establish that this effect can be reconstructed at the level of a single nucleosome. H1 selectively represses oocyte-type 5S rRNA genes by directing the stable positioning of a nucleosome such that transcription factors cannot bind to the gene. This effect does not occur on the somatic-type genes. Histone H1 binds to the 5′ end of the nucleosome core on the somatic 5S rRNA gene, leaving key regulatory elements in the promoter accessible, while histone H1 binds to the 3′ end of the nucleosome core on the oocyte 5S rRNA genes, specifically blocking access to a key promoter element (the C box). TFIIIA can bind to the somatic 5S rRNA gene assembled into a nucleosome in the presence of H1. Because H1 binds with equivalent affinities to nucleosomes containing either gene, we establish that it is the sequence-selective assembly of a specific repressive chromatin structure on the oocyte 5S rRNA genes that accounts for differential transcriptional repression. Thus, general components of chromatin can determine the assembly of specific regulatory nucleoprotein complexes.

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Genomic environments scale the activities of diverse core promoters

Genome Research ◽

10.1101/gr.276025.121 ◽

2021 ◽

Author(s):

Clarice K.Y. Hong ◽

Barak A. Cohen

Keyword(s):

Core Promoter ◽

Classical Model ◽

Regulatory Elements ◽

Sequence Motifs ◽

Core Promoters ◽

The Core ◽

Genomic Location ◽

Basal Transcriptional Machinery ◽

Promoter Specificity ◽

Genomic Locations

A classical model of gene regulation is that enhancers provide specificity whereas core promoters provide a modular site for the assembly of the basal transcriptional machinery. However, examples of core promoter specificity have led to an alternate hypothesis in which specificity is achieved by core promoters with different sequence motifs that respond differently to genomic environments containing different enhancers and chromatin landscapes. To distinguish between these models, we measured the activities of hundreds of diverse core promoters in four different genomic locations and, in a complementary experiment, six different core promoters at thousands of locations across the genome. Although genomic locations had large effects on expression, the intrinsic activities of different classes of promoters were preserved across genomic locations, suggesting that core promoters are modular regulatory elements whose activities are independently scaled up or down by different genomic locations. This scaling of promoter activities is nonlinear and depends on the genomic location and the strength of the core promoter. Our results support the classical model of regulation in which diverse core promoter motifs set the intrinsic strengths of core promoters, which are then amplified or dampened by the activities of their genomic environments.

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